DNA knock-in system

ABSTRACT

The present disclosure relates to an efficient genome editing technique. In one aspect, the technique can greatly improve the efficiency of homologous recombination during intracellular targeting, including gene targeting. Using this technique, genetically modified cell lines, rat, mouse, zebrafish, and fertilized eggs of other species can be quickly and efficiently generated.

RELATED APPLICATIONS

This application is a National Phase application under 35 U.S.C. § 371of International Application No. PCT/US2015/045134 filed on Aug. 13,2015, which claims priority benefit to U.S. Provisional PatentApplication No. 62/037,551, filed on Aug. 14, 2014, the contents ofwhich are incorporated herein by reference in their entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 735782000100SEQUST.TXT,date recorded: Feb. 13, 2017, size: 24 KB).

TECHNICAL FIELD

The present invention relates to an enhanced DNA knock-in (EKI) systemand uses thereof.

BACKGROUND

Recent developments in DNA manipulation allows for effectiveintroduction of exogenous genes into the chromosome of a host cell (geneknock-in). This allows the insertion of a protein coding cDNA sequenceat a particular locus in an organism's genome, for example, insertion ofa mutation or exogenous gene at a particular locus on a chromosome. Forexample, a point mutation can be introduced into a target gene byknock-in to model human genetic disorders. In addition, exogenous genessuch as reporter genes (EGFP, mRFP, mCherry, tdTomato etc.) can beintroduced by homologous recombination into a particular locus of thetarget gene, and can be used to track expression of the target gene andstudy its expression profiles by expression of the reporter gene.

In many circumstances, gene knock-in involves homologous recombinationmechanisms in an organism. Under natural circumstances, the probabilityof homologous recombination between an exogenous targeting vector andthe genome of a cell is very low, about 1/10⁵ to 1/10⁶. Spontaneous genetargeting typically occurs at a very low frequency in mammalian cellswith an efficiency of 1 in a million cells. The presence of adouble-strand break is often recombinogenic and increases the homologousrecombination frequency by several thousand folds. See Jasin, 1996,“Genetic manipulation of genomes with rare-cutting endonucleases,”Trends in genetics: TIG 12(6): 224-228. In plants, the generation of adouble-strand break in DNA is known to increase the frequency ofhomologous recombination from a background level of about 10⁻³-10⁻⁴ by afactor of approximately 100-fold. See Hanin et al., 2001, “Genetargeting in Arabidopsis,” Plant J. 28:671-77. Generation of geneticallymodified mice via homologous recombination was made possible by theestablishment of murine embryonic stem cell lines. For example,targeting vectors can be constructed using bacterial artificialchromosome (BAC), and introduced into murine embryonic stem cells viatransfection (e.g., electroporation). Positive embryonic stem cellclones are selected and injected into mouse blastocysts microscopic cellmass, and then implanted into a surrogate mouse to produce geneticallyengineered chimeric mouse. However, methods for establishing embryonicstem cell lines from other species have not been as successful andwidely used.

Recently developed techniques, including ZFNs (zinc finger nucleases),TALENs (transcription activator-like effector nucleases), CRISPR(clustered regularly interspaced short palindromic repeats)/Cas9, andother site-specific nuclease technologies, made it possible to createdouble-strand DNA breaks at desired locus sites. These controlleddouble-strand breaks can promote homologous recombination at suchspecific locus sites. This process relies on targeting specificsequences of nucleic acid molecules, such as chromosomes, withendonucleases that recognize and bind to such sequences and induce adouble-strand break in the nucleic acid molecule. The double-strandbreak is repaired either by an error-prone nonhomologous end-joining(NHEJ) or by homologous recombination (HR).

Homologous recombination that occurs during DNA repair tends to resultin non-crossover products, in effect restoring the damaged DNA moleculeas it existed before the double-strand break, or generating arecombinant molecule by incorporating sequence(s) of a template. Thelatter has been used in gene targeting, protein engineering, and genetherapy. If the template for homologous recombination is provided intrans (e.g., by introducing an exogenous template into a cell), thedouble-strand break in the cell can be repaired using the providedtemplate. In gene targeting, the initial double-strand break increasesthe frequency of targeting by several orders of magnitude, compared toconventional homologous recombination-based gene targeting. Inprinciple, this method could be used to insert any sequence at the siteof repair so long as it is flanked by appropriate regions homologous tothe sequences near the double-strand break. Although this method has hadsuccess in various species such as mice and rats, the efficiency andsuccess rate of homologous recombination remain low, preventing themethod from being widely used. For instance, the method remains costlyand technically challenging for both scientific and commercial use.

The disclosures of all publications, patents, patent applications andpublished patent applications referred to herein are hereby incorporatedherein by reference in their entirety.

BRIEF SUMMARY OF THE INVENTION

In one embodiment, disclosed herein is a method of inserting a donorsequence at a predetermined insertion site on a chromosome in aneukaryotic cell, comprising: (a) introducing into the cell asequence-specific nuclease that cleaves the chromosome at the insertionsite; (b) introducing into the cell a donor construct; and (c)introducing into the cell an exonuclease. In one aspect, the donorconstruct is a linear nucleic acid or cleaved within the cell to producea linear nucleic acid. In another aspect, the linear nucleic acidcomprises a 5′ homology arm, the donor sequence, and a 3′ homology arm.In certain aspects, the 5′ homology arm is homologous to a sequenceupstream of the nuclease cleavage site on the chromosome, and the 3′homology arm is homologous to a sequence downstream of the cleavage siteon the chromosome. In some embodiments, the 5′ homology arm and the 3′homology arm are proximal to the 5′ and 3′ ends of the linear nucleicacid, respectively. In other embodiments, the donor sequence is insertedinto the chromosome at the insertion site through homologousrecombination.

In some embodiments, the sequence-specific nuclease used herein is azinc finger nuclease (ZFN). In some embodiments, the sequence-specificnuclease used herein is a transcription activator-like effector nuclease(TALEN). In some embodiments, the sequence-specific nuclease used hereinis an RNA-guided nuclease. In one aspect, the RNA-guided nuclease isCas, for example, Cas9.

In any of the preceding embodiments involving an RNA-guided nuclease,the method can further comprise introducing into the cell a guide RNA(gRNA) recognizing the insertion site.

In any of the preceding embodiments, the sequence-specific nuclease canbe introduced into the cell as a protein, mRNA, or cDNA.

In some embodiments, the sequence homology between the 5′ homology armand the sequence 5′ to the insertion site is at least about 80%. In someembodiments, the sequence homology between the 3′ homology arm and thesequence 3′ to the insertion site is at least about 80%. In any of thepreceding embodiments, the 5′ homology arm and the 3′ homology arm canbe at least about 200 base pair (bp).

In any of the preceding embodiments, the exonuclease can be a 5′ to 3′exonuclease. In one aspect, the exonuclease is a herpes simplex virustype 1 (HSV-1) exonuclease. In one embodiment, the exonuclease is UL12.

In any of the preceding embodiments, the donor construct can be a linearnucleic acid. In some embodiments, the donor construct is circular whenintroduced into the cell and cleaved within the cell to produce a linearnucleic acid. In one aspect, the donor construct further comprises a 5′flanking sequence upstream of the 5′ homology arm and a 3′ flankingsequence downstream of the 3′ homology arm. In one embodiment, the 5′flanking sequence or the 3′ flanking sequence is about 1 to about 500bp.

In some embodiments, a method disclosed herein further comprisesintroducing into the cell a second sequence-specific nuclease thatcleaves the donor construct at one or both of the flanking sequences,thereby producing the linear nucleic acid. In certain embodiments, thesequence-specific nuclease is an RNA-guided nuclease, and the methodfurther comprises introducing into the cell a second guide RNArecognizing one or both of the flanking sequences.

In any of the preceding embodiments, the eukaryotic cell can be amammalian cell. In some embodiments, the mammalian cell is a zygote or apluripotent stem cell.

In some aspects, disclosed herein is a method of generating agenetically modified animal, which comprises a donor sequence insertedat a predetermined insertion site on the chromosome of the animal. Inone embodiment, the method comprises: (a) introducing into a cell asequence-specific nuclease that cleaves the chromosome at the insertionsite; (b) introducing into the cell a donor construct; (c) introducinginto the cell an exonuclease; and (d) introducing the cell into acarrier animal to produce the genetically modified animal. In oneembodiment, the donor construct is a linear nucleic acid or cleavedwithin the cell to produce a linear nucleic acid. In one aspect, thelinear nucleic acid comprises a 5′ homology arm, the donor sequence, anda 3′ homology arm. In one aspect, the 5′ homology arm is homologous to asequence upstream of the nuclease cleavage site on the chromosome. Inone aspect, the 3′ homology arm is homologous to a sequence downstreamof the cleavage site on the chromosome. In some embodiments, the 5′homology arm and the 3′ homology arm are proximal to the 5′ and 3′ endsof the linear nucleic acid, respectively. In some embodiments, the donorsequence is inserted into the chromosome at the insertion site throughhomologous recombination.

In one aspect, the genetically modified animal is a rodent. In otheraspects, the cell is a zygote or a pluripotent stem cell.

In some aspects, provided herein is a genetically modified animalgenerated by the method of any of the methods described above.

In other aspects, provided herein is a kit for inserting a donorsequence at an insertion site on a chromosome in an eukaryotic cell,comprising: (a) a sequence-specific nuclease that cleaves the chromosomeat the insertion site; (b) a donor construct; and (c) an exonuclease. Inone embodiment, the donor construct is a linear nucleic acid or can becleaved within a cell to produce a linear nucleic acid. In one aspect,the linear nucleic acid comprises a 5′ homology arm, the donor sequence,and a 3′ homology arm. In one aspect, the 5′ homology arm is homologousto a sequence upstream of the nuclease cleavage site on the chromosome.In one aspect, the 3′ homology arm is homologous to a sequencedownstream of the cleavage site on the chromosome. In one aspect, the 5′homology arm and the 3′ homology arm are proximal to the 5′ and 3′ endsof the linear nucleic acid, respectively. In some embodiments, thesequence-specific nuclease is an RNA-guided nuclease. In otherembodiments, the kit further comprises a guide RNA (gRNA) recognizingthe insertion site.

In some embodiments, the donor construct is circular. In one aspect, thedonor construct further comprises a 5′ flanking sequence upstream of the5′ homology arm and a 3′ flanking sequence downstream of the 3′ homologyarm. In another aspect, the 5′ flanking sequence or the 3′ flankingsequence is about 1 to about 500 bp. In any of the precedingembodiments, the sequence-specific nuclease can be an RNA-guidednuclease, and the kit can further comprise a second guide RNArecognizing one or both of the flanking sequences. In any of thepreceding embodiments, the exonuclease can be a 5′ to 3′ exonuclease. Inone aspect, the exonuclease is a herpes simplex virus type 1 (HSV-1)exonuclease. In another aspect, the exonuclease is UL12. In oneembodiment, the UL12 is fused to a nuclear localization sequence (NLS).

In some embodiments, the present disclosure provides a method ofproducing a linear nucleic acid in an eukaryotic cell, in which thelinear nucleic acid comprises a 5′ homology arm, a donor sequence, and a3′ homology arm; the 5′ homology arm is homologous to a sequenceupstream of the nuclease cleavage site on the chromosome and the 3′homology arm is homologous to a sequence downstream of the cleavage siteon the chromosome; and the 5′ homology arm and the 3′ homology arm areproximal to the 5′ and 3′ ends of the linear nucleic acid, respectively.In one aspect, the method comprises: (a) introducing into the cell acircular donor construct comprising the linear nucleic acid and furthercomprising a 5′ flanking sequences upstream of the 5′ homology arm and a3′ flanking sequence downstream of the 3′ homology arm; and (b)introducing into the cell a sequence-specific nuclease, wherein thesequence-specific nuclease cleaves the circular donor construct at the5′ flanking sequence and the 3′ flanking sequence, thereby producing thelinear nucleic acid. In one aspect, the sequence-specific nuclease isZFN. In another aspect, the sequence-specific nuclease is TALEN. In yetanother aspect, the sequence-specific nuclease is an RNA-guidednuclease. In one embodiment, the RNA-guided nuclease is Cas. In someembodiments, the RNA-guided nuclease is Cas9. In any of the precedingembodiments, the method can further comprise introducing into the cell aguide RNA recognizing the 5′ flanking sequence and/or the 3′ flankingsequence.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts a targeting scheme for knocking in EGFP-ACTB in the U2OScell line.

FIG. 2 shows fluorescent microscopic images indicating the expression ofthe EGFP-ACTB fusion protein in U2OS cells after knocking in using theenhanced knock-in (“EKI”) system.

FIGS. 3A, 3B AND 3C depict flow cytometry results showing that theknock-in efficiency of EGFP-ACTB in U2OS cells was significantlyincreased using the EKI system, compared to conventionalCRISPR/Cas9-mediated knock-in. FIG. 3A shows the result of a controlexperiment, FIG. 3B shows the result of conventionalCRISPR/Cas9-mediated knock-in, and FIG. 3C shows the result of the EKIsystem-mediated knock-in.

FIG. 4 depicts a targeting scheme for knocking in EGFP-LMNB1 in the C6cell line.

FIG. 5 shows fluorescent microscopic images indicating the expression ofthe EGFP-LMNB1 fusion protein in C6 cells after knocking in using theEKI system.

FIGS. 6A, 6B AND 6C depict flow cytometry results showing that theknock-in efficiency of EGFP-LMNB1 in C6 cells was significantlyincreased using the EKI system, compared to conventionalCRISPR/Cas9-mediated knock-in. FIG. 6A shows the result of a controlexperiment, FIG. 6B shows the result of conventionalCRISPR/Cas9-mediated knock-in, and FIG. 6C shows the result of the EKIsystem-mediated knock-in.

FIGS. 7A AND 7B depict a targeting scheme for double knock-in ofEGFP-ACTB and mCherry-LMNB1 in the U2OS cell line. FIG. 7A depicts thetargeting scheme for knocking in EGFP-ACTB, and FIG. 7B depicts thetargeting scheme for knocking in mCherry-LMNB1.

FIG. 8 shows fluorescent microscopic images indicating successful doubleknock-in and expression of the EGFP-ACTB and mCherry-LMNB1 fusionproteins in the same U2OS cell mediated by the EKI system.

FIG. 9 depicts a targeting scheme for generating CD4-2A-dsRed knock-inrats.

FIG. 10 depicts genotyping results of CD4-2A-dsRed knock-in rats of theF0 generation, using 5′-junction PCR reaction.

FIG. 11 depicts genotyping results of CD4-2A-dsRed knock-in rats of theF0 generation, using 3′-junction PCR reaction.

FIGS. 12A AND 12B depict southern blot results for the CD4-2A-dsRedknock-in rats. FIG. 12A shows the southern blot strategy, and FIG. 12Bshows the southern blot results using the 5′ probe, 3′ probe, and dsRedprobe. The two F1 rats (#19 and #21) tested with southern blots were theoffspring of the #22 F0 rat in FIG. 10 and FIG. 11. WT: wild-type rat.PC: positive control.

FIG. 13 depicts a targeting scheme for knocking in TH-GFP in the H9 cellline.

FIG. 14 shows a fluorescent microscopic image indicating the expressionof the TH-GFP fusion protein in H9 cells after knocking in using the EKIsystem.

FIG. 15 shows the locations of the genotyping primers for TH-GFPknock-in in H9 cells. LA: left homology arm. RA: right homology arm.

FIGS. 16A AND 16B depict genotyping results of TH-GFP knock-in in H9cells. FIG. 16A depicts the 5′-junction PCR reaction results, and FIG.16B depicts the 3′-junction PCR reaction results. WT: wild-type H9cells.

FIG. 17 shows the sequencing results of the PCR products from H9-TH-GFPcell line #1 in FIG. 16. Shown in the chromatogram are sequences ofCAGACGTACCAGTCAGTCTACTTCGTGTCTGAGAGCTTCAGTGACG (SEQ ID NO: 40) andCTCCTCTCAAGGAGGCACCCATGTCCTCTCCAGCTGCCGGGCCTCA (SEQ ID NO: 41).

FIG. 18 shows the karyotype of H9-TH-GFP cell line #1 from FIG. 16.

FIG. 19 depicts a targeting scheme for knocking in OCT4-EGFP in the H9cell line.

FIG. 20 shows a fluorescent microscopic image indicating the expressionof the OCT4-EGFP fusion protein in H9 cells after knocking in using theEKI system.

FIG. 21 shows the locations of the genotyping primers for OCT4-EGFPknock-in in H9 cells. LA: left homology arm. RA: right homology arm.

FIGS. 22A, 22B AND 22C depict genotyping results of OCT4-EGFP knock-inin H9 cells. FIG. 22A depicts the 5′-junction PCR reaction results, FIG.22B depicts the 3′-junction PCR reaction results, and FIG. 22C depictsthe full length PCR reaction results. wt: wild-type H9 cells. c-: H₂O asnegative control.

FIG. 23 shows the sequencing results of the PCR products fromH9-OCT4-EGFP cell line #6 in FIG. 22. Shown in the chromatogram aresequences of GATACCCGGGGACCTTCCCTTTCTTGGCCTAATTTCCATTGCTTC (SEQ ID NO:42) and GTGGGTTAAGCGGTTTGATTCACACTGAACCAGGCCAGCCCAGTTG (SEQ ID NO: 43).

FIG. 24 shows the karyotype of H9-OCT4-EGFP cell line #6 in FIG. 22.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a novel DNA knock-in method which allowsfor the introduction of one or more exogenous sequences into a specifictarget site on the cellular chromosome with significantly higherefficiency compared to traditional DNA knock-in methods usingsequence-specific nucleases such as CRISPR/Cas9 or TALEN-based geneknock-in systems. In addition to the use of a sequence-specificnuclease, the method of the present application further utilizes anexonuclease (such as a 5′ to 3′ exonuclease, for example UL12) inconjunction with a donor construct which is either a linear nucleic acidor can be cleaved within the cell to produce a linear nucleic acid. TheDNA knock-in system allows donor sequences to be inserted at any desiredtarget site with high efficiency, making it feasible for many uses suchas creation of transgenic animals expressing exogenous genes, modifying(e.g., mutating) a genomic locus, and gene editing, for example byadding an exogenous non-coding sequence (such as sequence tags orregulatory elements) into the genome. The cells and animals producedusing methods provided herein can find various applications, for exampleas cellular therapeutics, as disease models, as research tools, and ashumanized animal useful for various purposes.

Thus, the present application in one aspect provides methods ofinserting a donor sequence at a predetermined insertion site on achromosome of an eukaryotic cell.

In another aspect, there are provided methods of generating a linearnucleic acid in a cell.

In another aspect, there is provided a kit for use in any one of themethods described herein.

In another aspect, there is provided a method of generating agenetically modified animal by using the gene knock-in system describedherein.

Reference to “about” a value or parameter herein includes (anddescribes) variations that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X”.

As used herein and in the appended claims, the singular forms “a”, “or”,and “the” include plural referents unless the context clearly dictatesotherwise.

The compositions and methods of the present invention may comprise,consist of, or consist essentially of the essential elements andlimitations of the invention described herein, as well as any additionalor optional ingredients, components, or limitations described herein orotherwise useful in a nutritional or pharmaceutical application.

Methods of the Present Invention

The present invention provides methods of inserting a donor sequence ata predetermined insertion site on a chromosome in an eukaryotic cell. Insome embodiments the method comprises: a) introducing into the cell asequence-specific nuclease that cleaves the chromosome at the insertionsite; b) introducing into the cell a donor construct; and c) introducinginto the cell an exonuclease; wherein the donor construct is a linearnucleic acid or cleaved within the cell to produce a linear nucleicacid, wherein the linear nucleic acid comprises a 5′ homology arm, thedonor sequence, and a 3′ homology arm, wherein the 5′ homology arm ishomologous to a sequence upstream of the nuclease cleavage site on thechromosome and wherein the 3′ homology arm is homologous to a sequencedownstream of the nuclease cleavage site on the chromosome; wherein the5′ homology arm and the 3′ homology arm are proximal to the 5′ and 3′ends of the linear nucleic acid, respectively, and wherein the donorsequence is inserted into the chromosome at the insertion site throughhomologous recombination.

In some embodiments, the sequence-specific nuclease, the exonuclease,and the donor construct are introduced into the cell simultaneously. Insome embodiments, at least one of the three components is introducedinto the cell at a different time from the other component(s). Forexample, the donor construct may be introduced into the cell first, andthe sequence-specific nuclease and the exonuclease are subsequentlyintroduced. In some embodiments, the sequence-specific nuclease isintroduced into the cell first, and the donor construct and theexonuclease are subsequently introduced. In some embodiments, all threecomponents are introduced at a different time point relative to eachother. For example, the three components can be administered in asequence, one after another at a specific order.

In some embodiments, the sequence-specific nuclease and/or theexonuclease are introduced into the cell as a cDNA. In some embodiments,the sequence-specific nuclease and/or the exonuclease are introducedinto the cell as an mRNA. In some embodiments, the sequence-specificnuclease and/or the exonuclease are introduced into the cell as aprotein.

For example, in some embodiments, there is provided a method ofinserting a donor sequence at a predetermined insertion site on achromosome in an eukaryotic cell, the method comprising: a) introducinginto the cell a nucleic acid sequence encoding a sequence-specificnuclease that cleaves the chromosome at the insertion site; b)introducing into the cell a donor construct; and c) introducing into thecell a nucleic acid sequence encoding an exonuclease; wherein the donorconstruct is a linear nucleic acid or cleaved within the cell to producea linear nucleic acid, wherein the linear nucleic acid comprises a 5′homology arm, the donor sequence, and a 3′ homology arm, wherein the 5′homology arm is homologous to a sequence upstream of the nucleasecleavage site on the chromosome and wherein the 3′ homology arm ishomologous to a sequence downstream of the nuclease cleavage site on thechromosome; wherein the 5′ homology arm and the 3′ homology arm areproximal to the 5′ and 3′ ends of the linear nucleic acid, respectively,and wherein the donor sequence is inserted into the chromosome at theinsertion site through homologous recombination. In some embodiments,the nucleic acid encoding the sequence-specific nuclease and/or thenucleic acid encoding the exonuclease is mRNA. In some embodiments, thenucleic acid encoding the sequence-specific nuclease and/or the nucleicacid encoding the exonuclease is cDNA. In some embodiments, the nucleicacid encoding the sequence-specific nuclease and/or the nucleic acidencoding the exonuclease is introduced into the cell by transfection(including for example transfection through electroporation). In someembodiments, the nucleic acid encoding the sequence-specific nucleaseand/or the nucleic acid encoding the exonuclease is introduced into thecell by injection.

In some embodiments, there is provided a method of inserting a donorsequence at a predetermined insertion site on a chromosome in aneukaryotic cell, the method comprising: a) introducing into the cell avector comprising a nucleic acid sequence encoding a sequence-specificnuclease that cleaves the chromosome at the insertion site; b)introducing into the cell a donor construct; and c) introducing into thecell a vector comprising a nucleic acid sequence encoding anexonuclease; wherein the donor construct is a linear nucleic acid orcleaved within the cell to produce a linear nucleic acid, wherein thelinear nucleic acid comprises a 5′ homology arm, the donor sequence, anda 3′ homology arm, wherein the 5′ homology arm is homologous to asequence upstream of the nuclease cleavage site on the chromosome andwherein the 3′ homology arm is homologous to a sequence downstream ofthe nuclease cleavage site on the chromosome; wherein the 5′ homologyarm and the 3′ homology arm are proximal to the 5′ and 3′ ends of thelinear nucleic acid, respectively, and wherein the donor sequence isinserted into the chromosome at the insertion site through homologousrecombination. In some embodiments, the vector comprising the nucleicacid encoding the sequence-specific nuclease and/or the vectorcomprising the nucleic acid encoding the exonuclease is introduced intothe cell by transfection (including for example transfection throughelectroporation).

In some embodiments, there is provided a method of inserting a donorsequence at a predetermined insertion site on a chromosome in aneukaryotic cell, the method comprising: a) introducing into the cell avector comprising a nucleic acid sequence encoding a sequence-specificnuclease that cleaves the chromosome at the insertion site and a nucleicacid sequence encoding an exonuclease; b) introducing into the cell adonor construct; wherein the donor construct is a linear nucleic acid orcleaved within the cell to produce a linear nucleic acid, wherein thelinear nucleic acid comprises a 5′ homology arm, the donor sequence, anda 3′ homology arm, wherein the 5′ homology arm is homologous to asequence upstream of the nuclease cleavage site on the chromosome andwherein the 3′ homology arm is homologous to a sequence downstream ofthe nuclease cleavage site on the chromosome; wherein the 5′ homologyarm and the 3′ homology arm are proximal to the 5′ and 3′ ends of thelinear nucleic acid, respectively, and wherein the donor sequence isinserted into the chromosome at the insertion site through homologousrecombination. In some embodiments, the vector comprising the nucleicacid encoding the sequence-specific nuclease and the nucleic acidencoding the exonuclease is introduced into the cell by transfection(including for example transfection through electroporation).

In some embodiments, there is provided a method of inserting a donorsequence at a predetermined insertion site on a chromosome in aneukaryotic cell (such as a zygotic cell), the method comprising: a)introducing (such as injecting) into the cell an mRNA sequence encodinga sequence-specific nuclease that cleaves the chromosome at theinsertion site; b) introducing (such as injecting) into the cell a donorconstruct; and c) introducing (such as injecting) into the cell an mRNAsequence encoding an exonuclease; wherein the donor construct is alinear nucleic acid or cleaved within the cell to produce a linearnucleic acid, wherein the linear nucleic acid comprises a 5′ homologyarm, the donor sequence, and a 3′ homology arm, wherein the 5′ homologyarm is homologous to a sequence upstream of the nuclease cleavage siteon the chromosome and wherein the 3′ homology arm is homologous to asequence downstream of the nuclease cleavage site on the chromosome;wherein the 5′ homology arm and the 3′ homology arm are proximal to the5′ and 3′ ends of the linear nucleic acid, respectively, and wherein thedonor sequence is inserted into the chromosome at the insertion sitethrough homologous recombination. In some embodiments, the introduction(such as injection) is carried out in vitro. In some embodiments, theintroduction (such as injection) is carried out in vivo. In someembodiments, the method further comprises transcribing in vitro anucleic acid encoding the sequence-specific nuclease into mRNA. In someembodiments, the method further comprises transcribing in vitro anucleic acid encoding the exonuclease into mRNA.

For example, in some embodiments, there is provided a method ofinserting a donor sequence at a predetermined insertion site on achromosome in an immune cell (such as T cells), the method comprising:a) introducing into the cell a nucleic acid sequence encoding asequence-specific nuclease that cleaves the chromosome at the insertionsite; b) introducing into the cell a donor construct; and c) introducinginto the cell a nucleic acid sequence encoding an exonuclease; whereinthe donor construct is a linear nucleic acid or cleaved within the cellto produce a linear nucleic acid, wherein the linear nucleic acidcomprises a 5′ homology arm, the donor sequence, and a 3′ homology arm,wherein the 5′ homology arm is homologous to a sequence upstream of thenuclease cleavage site on the chromosome and wherein the 3′ homology armis homologous to a sequence downstream of the nuclease cleavage site onthe chromosome; wherein the 5′ homology arm and the 3′ homology arm areproximal to the 5′ and 3′ ends of the linear nucleic acid, respectively,and wherein the donor sequence is inserted into the chromosome at theinsertion site through homologous recombination. In some embodiments,the nucleic acid encoding the sequence-specific nuclease and/or thenucleic acid encoding the exonuclease is mRNA. In some embodiments, thenucleic acid encoding the sequence-specific nuclease and/or the nucleicacid encoding the exonuclease is cDNA. In some embodiments, the nucleicacid encoding the sequence-specific nuclease and/or the nucleic acidencoding the exonuclease is introduced into the cell by transfection(including for example transfection through electroporation). In someembodiments, the nucleic acid encoding the sequence-specific nucleaseand/or the nucleic acid encoding the exonuclease is introduced into thecell by injection.

In some embodiments, there is provided a method of inserting a donorsequence at a predetermined insertion site on a chromosome in an immunecell (such as T cells), the method comprising: a) introducing into thecell a vector comprising a nucleic acid sequence encoding asequence-specific nuclease that cleaves the chromosome at the insertionsite; b) introducing into the cell a donor construct; and c) introducinginto the cell a vector comprising a nucleic acid sequence encoding anexonuclease; wherein the donor construct is a linear nucleic acid orcleaved within the cell to produce a linear nucleic acid, wherein thelinear nucleic acid comprises a 5′ homology arm, the donor sequence, anda 3′ homology arm, wherein the 5′ homology arm is homologous to asequence upstream of the nuclease cleavage site on the chromosome andwherein the 3′ homology arm is homologous to a sequence downstream ofthe nuclease cleavage site on the chromosome; wherein the 5′ homologyarm and the 3′ homology arm are proximal to the 5′ and 3′ ends of thelinear nucleic acid, respectively, and wherein the donor sequence isinserted into the chromosome at the insertion site through homologousrecombination. In some embodiments, the vector comprising the nucleicacid encoding the sequence-specific nuclease and/or the vectorcomprising the nucleic acid encoding the exonuclease is introduced intothe cell by transfection (including for example transfection throughelectroporation).

In some embodiments, there is provided a method of inserting a donorsequence at a predetermined insertion site on a chromosome in an immunecell (such as T cells), the method comprising: a) introducing into thecell a vector comprising a nucleic acid sequence encoding asequence-specific nuclease that cleaves the chromosome at the insertionsite and a nucleic acid sequence encoding an exonuclease; b) introducinginto the cell a donor construct; wherein the donor construct is a linearnucleic acid or cleaved within the cell to produce a linear nucleicacid, wherein the linear nucleic acid comprises a 5′ homology arm, thedonor sequence, and a 3′ homology arm, wherein the 5′ homology arm ishomologous to a sequence upstream of the nuclease cleavage site on thechromosome and wherein the 3′ homology arm is homologous to a sequencedownstream of the nuclease cleavage site on the chromosome; wherein the5′ homology arm and the 3′ homology arm are proximal to the 5′ and 3′ends of the linear nucleic acid, respectively, and wherein the donorsequence is inserted into the chromosome at the insertion site throughhomologous recombination. In some embodiments, the vector comprising thenucleic acid encoding the sequence-specific nuclease and the nucleicacid encoding the exonuclease is introduced into the cell bytransfection (including for example transfection throughelectroporation).

In some embodiments, there is provided a method of inserting a donorsequence at a predetermined insertion site on a chromosome in an immunecell (such as T cells), the method comprising: a) introducing (such asinjecting) into the cell an mRNA sequence encoding a sequence-specificnuclease that cleaves the chromosome at the insertion site; b)introducing (such as injecting) into the cell a donor construct; and c)introducing (such as injecting) into the cell an mRNA sequence encodingan exonuclease; wherein the donor construct is a linear nucleic acid orcleaved within the cell to produce a linear nucleic acid, wherein thelinear nucleic acid comprises a 5′ homology arm, the donor sequence, anda 3′ homology arm, wherein the 5′ homology arm is homologous to asequence upstream of the nuclease cleavage site on the chromosome andwherein the 3′ homology arm is homologous to a sequence downstream ofthe nuclease cleavage site on the chromosome; wherein the 5′ homologyarm and the 3′ homology arm are proximal to the 5′ and 3′ ends of thelinear nucleic acid, respectively, and wherein the donor sequence isinserted into the chromosome at the insertion site through homologousrecombination. In some embodiments, the introduction (such as injection)is carried out in vitro. In some embodiments, the introduction (such asinjection) is carried out in vivo. In some embodiments, the methodfurther comprises transcribing in vitro a nucleic acid encoding thesequence-specific nuclease into mRNA. In some embodiments, the methodfurther comprises transcribing in vitro a nucleic acid encoding theexonuclease into mRNA.

The cells described herein can be any eukaryotic cell, e.g., an isolatedcell of an animal, such as a totipotent, pluripotent, or adult stemcell, a zygote, or a somatic cell. In some embodiments, the cell is froma primary cell culture. In some embodiments, cells for use in themethods are human cells. In some embodiments, cells for use in themethods are yeast cells. In some embodiments, the cell is from adomesticated animal (e.g., cow, sheep, cat, dog, and horse). In someembodiments, the cell is from a primate (e.g., non-human primate such asmonkey). In some embodiments, the cell is from a rabbit. In someembodiments, the cell is from a fish (such as zebrafish). In someembodiments, the cell is from a rodent (e.g., mouse, rat, hamster,guinea pig). In some embodiments, the cell is from a non-vertebrate(e.g., Drosophila melanogaster and Caenorhabditis elegans).

The cells described herein can be immune cells, which include, but arenot limited to, granulocytes (such as basophils, eosinophils, andneutrophils), mast cells, monocytes, dendritic cells (DC), naturalkiller (NK) cells, B cells, and T cells (such as CD8+ T cells, and CD4+T cells). In some embodiments, the cell is a CD4+ T cell (e.g., T helpercells TH1, TH2, TH17, and regulatory T cells).

The methods and compositions described herein can be used to insert adonor sequence into one or more genomic locus in the cell. In certainembodiments, the methods and compositions described herein can be usedto target more than one genomic locus within a cell, e.g., for doubleDNA knock-in. In certain embodiments, the methods and compositionsdescribed herein are used to target two, three, four, five, six, seven,eight, nine, ten, or more than ten genomic loci within a cell. In someaspects, the double or multiple knock-in can be carried outsimultaneously or sequentially. For example, the reagents for targetingthe two or more genomic loci within the same cell are mixed andintroduced into the cell at substantially the same time. In otherembodiments, reagents that target each of the multiple genomic loci canbe introduced into the cell in a sequence, one after another at aspecific order. In yet other embodiments, a first genomic locus istargeted and cells with successful knock-in are selected, enriched,and/or separated, and are subjected to targeting a second genomic locus.

Double or multiple knock-in can be accomplished, for example, by usingtwo or more different sequence-specific nucleases, each recognizing asequence at one of the predetermined insertion sites. Thesesequence-specific nucleases can be introduced into a cell simultaneouslyor sequentially. Thus, for example, in some embodiments, there isprovided a method of inserting two or more donor sequences, each at apredetermined insertion sites on a chromosome in an eukaryotic cell,comprising: a) introducing into the cell one or more sequence-specificnucleases that cleave the chromosome at the predetermined insertionsites; b) introducing into the cell two or more donor constructs; and c)introducing into the cell an exonuclease, wherein each of the donorconstruct is a linear nucleic acid or cleaved within the cell to producea linear nucleic acid, wherein the linear nucleic acid comprises a 5′homology arm, the donor sequence, and a 3′ homology arm, wherein the 5′homology arm is homologous to a sequence upstream of the correspondingnuclease cleavage site on the chromosome, and wherein the 3′ homologyarm is homologous to a sequence downstream of the corresponding nucleasecleavage site on the chromosome, wherein the 5′ homology arm and the 3′homology arm are proximal to the 5′ and 3′ ends of the linear nucleicacid, respectively, and wherein the two or more donor sequences areinserted into the chromosome at the predetermined insertion sitesthrough homologous recombination. In some embodiments, there is provideda method of inserting two donor sequences, each at a predeterminedinsertion site on a chromosome in an eukaryotic cell, comprising: a)introducing into the cell a first sequence-specific nuclease thatcleaves the chromosome at a first predetermined insertion site; b)introducing into the cell a first donor construct; c) introducing intothe cell a second sequence-specific nuclease that cleaves the chromosomeat a second predetermined insertion site; and d) introducing into thecell a second donor construct; and e) introducing into the cell anexonuclease, wherein each of the donor construct is a linear nucleicacid or cleaved within the cell to produce a linear nucleic acid,wherein the linear nucleic acid comprises a 5′ homology arm, the donorsequence, and a 3′ homology arm, wherein the 5′ homology arm ishomologous to a sequence upstream of the corresponding nuclease cleavagesite on the chromosome, and wherein the 3′ homology arm is homologous toa sequence downstream of the corresponding nuclease cleavage site on thechromosome, wherein the 5′ homology arm and the 3′ homology arm areproximal to the 5′ and 3′ ends of the linear nucleic acid, respectively,and wherein the two donor sequences are inserted into the chromosome atthe predetermined insertion sites through homologous recombination.

The sequence-specific nuclease (and exonuclease described herein) can beintroduced into the cell in form of a protein or in form of a nucleicacid encoding the sequence-specific nuclease (and exonuclease describedherein), such as an mRNA or a cDNA. Nucleic acids can be delivered aspart of a larger construct, such as a plasmid or viral vector, ordirectly, e.g., by electroporation, lipid vesicles, viral transporters,microinjection, and biolistics. For example, the sequence-specificnuclease (and exonuclease described herein) can be introduced into thecell by a variety of means known in the art, including transfection,calcium phosphate-DNA co-precipitation, DEAE-dextran-mediatedtransfection, polybrene-mediated transfection, electroporation,microinjection, transduction, cell fusion, liposome fusion, lipofection,protoplast fusion, retroviral infection, use of a gene gun, use of a DNAvector transporter, and biolistics (e.g., particle bombardment) (Seee.g., Wu et al., 1992, J. Biol. Chem., 267:963-967; Wu and Wu, 1988, J.Biol. Chem., 263:14621-14624; and Williams et al., 1991, Proc. Natl.Acad. Sci. USA 88:2726-2730). Receptor-mediated DNA delivery approachescan also be used (Curiel et al., 1992, Hum. Gene Ther., 3:147-154; andWu and Wu, 1987, J. Biol. Chem., 262:4429-4432).

The donor construct can be introduced into the cell in the form of alinear nucleic acid or cleaved within the cell to produce a linearnucleic acid. It can be delivered by any method appropriate forintroducing nucleic acids into a cell. For example, the donor constructcan be introduced into the cell by a variety of means known in the art,including transfection, calcium phosphate-DNA co-precipitation,DEAE-dextran-mediated transfection, polybrene-mediated transfection,electroporation, microinjection, transduction, cell fusion, liposomefusion, lipofection, protoplast fusion, retroviral infection, use of agene gun, use of a DNA vector transporter, and biolistics (e.g.,particle bombardment) (See e.g., Wu et al., 1992, J. Biol. Chem.,267:963-967; Wu and Wu, 1988, J. Biol. Chem., 263:14621-14624; andWilliams et al., 1991, Proc. Natl. Acad. Sci. USA 88:2726-2730).Receptor-mediated DNA delivery approaches can also be used (Curiel etal., 1992, Hum. Gene Ther., 3:147-154; and Wu and Wu, 1987, J. Biol.Chem., 262:4429-4432).

In one embodiment, a target cell can be transfected with a nucleic acidcontaining a specific gene that leads to the expression of a geneproduct in the target cell, for example, a sequence-specific nuclease orexonuclease described herein. In another embodiment, a functionalprotein, e.g., a sequence-specific nuclease or exonuclease, is deliveredinto a target cell, using membrane-disrupting, pore-forming methods orreagents, such as micro-injection and electroporation, or other reagentssuch as liposomes as a carrier to deliver the protein across the cellmembrane. Using a variety of assays known in the art, introduction ofnucleic acids or proteins in the target cell can be confirmed and theireffects on cellular physiology and/or gene expression can be studied.

In some aspects, delivery of the sequence-specific nuclease, donorconstruct, and/or exonuclease described herein into a target cell isnonspecific, e.g., anything can enter or exit the cell once the membraneis disrupted. In other aspects, the delivery of nucleic acids and/orproteins into a target cell is specific. For example, thesequence-specific nuclease, donor construct, and/or exonucleasedescribed herein can be delivered into a cell using protein-transductiondomains (PTDs) and/or membrane-translocating peptides that mediateprotein delivery into cells. These PTDs or signal peptide sequences arenaturally occurring polypeptides of 15 to 30 amino acids, which normallymediate protein secretion in the cells. They are composed of apositively charged amino terminus, a central hydrophobic core and acarboxyl-terminal cleavage site recognized by a signal peptidase. Incertain embodiments, polypeptides, protein domains, and full-lengthprotein, including antibodies, can be introduced into cells usingsolution-based protein transfection protocols. In one aspect, theprotein to be introduced into cells is pre-complexed with a carrierreagent. In another embodiment, a fusion protein between the protein tobe introduced and another moiety is used. For example, the fusionprotein contains a protein (e.g., a sequence-specific nuclease and/orexonuclease) or a protein domain of interest, fused covalently with aprotein or peptide that exhibits properties for spontaneousintracellular penetration. Examples of such membrane-transducingpeptides include Trojan peptides, human immuodeficiency virus (HIV)-1transcriptional activator (TAT) protein or its functional domainpeptides, and other peptides containing protein-transduction domains(PTDs) derived from translocation proteins such as Drosophila homeotictranscription factor Antennapedia (Antp) and herpes simplex virusDNA-binding protein, VP22, and the like. Some commercially availablepeptides, for example, penetratin 1, Pep-1 (Chariot reagent, ActiveMotif Inc., CA) and HIV GP41 fragment (519-541), can be used.

In some embodiments, the exonuclease described herein is an alkalineexonuclease. In some embodiments, the exonuclease is a pH dependentalkaline exonuclease. In some embodiments, the exonuclease interactswith single-strand DNA binding protein and promotes strand exchange. Insome embodiments, the exonuclease is also an endonuclease. In someembodiments, the exonuclease is a 5′ to 3′ exonuclease. In someembodiments, the exonuclease is a herpes simplex virus-type 1 (HSV-1)exonuclease. In some embodiments, the exonuclease is UL-12, e.g., UL-12protein (SEQ ID NO: 2, accession number NP_044613.1) encoded by SEQ IDNO: 1 (accession number NC_001806.1, Gene ID: 2703382) as described inU.S. Pat. No. 7,135,324 B2, the disclosure of which is incorporatedherein in its entirety for all purposes. In some embodiments, theexonuclease is a UL-12 homolog from Epstaine-Barr virus, bovineherpesvirus type 1, pseudoorabies virus, and human cytomegalovirus(HCMV).

In herpes simplex virus, the HSV-1 alkaline nuclease UL-12 and the HSV-1single-strand DNA binding polypeptide (encoded by the ICP 8 gene andhereinafter referred to as “ICP8”; also known in the art as UL-29) worktogether to effect DNA strand exchange. As used herein, UL-12 refers toHSV-1 UL-12 as well as its homologs, orthologs, and paralogs. “Homolog”is a generic term used in the art to indicate a polynucleotide orpolypeptide sequence possessing a high degree of sequence relatedness toa subject sequence. Such relatedness may be quantified by determiningthe degree of identity and/or similarity between the sequences beingcompared. Falling within this generic term are the terms “ortholog”,meaning a polynucleotide or polypeptide that is the functionalequivalent of a polynucleotide or polypeptide in another species, and“paralog” meaning a functionally similar sequence when considered withinthe same species. Paralogs present in the same species or orthologs ofthe UL-12 gene in other species can readily be identified without undueexperimentation, by molecular biological techniques well known in theart.

Goldstein and Weller (1998, Virology, 244(2):442-57) examined theregions of HSV-1 UL-12 that are highly conserved among herpesvirushomologs and identified seven conserved amino acids regions. The sevenregions of homology among herpesviruses were originally reported inMartinez et al., 1996, Virology, 215:152-64. Baculoviruses also encode ahomolog of this protein (Ahrens et al., 1997, Virology, 229(2):381-99;Ayres et al., 1994, 202:586-605); however, only motifs I-IV are presentin these homologs.

The seven conserved motifs of HSV-1 UL-12 are as follows: Motif I (fromamino acid residue 218 to residue 244 of SEQ ID NO: 2), Motif II (fromamino acid residue 325 to residue 340 of SEQ ID NO: 2), Motif III (fromamino acid residue 362 to residue 377 of SEQ ID NO: 2), Motif IV (fromamino acid residue 415 to residue 445 of SEQ ID NO: 2), Motif V (fromamino acid residue 455 to residue 465 of SEQ ID NO: 2), Motif VI (fromamino acid residue 491 to residue 514 of SEQ ID NO: 2), and Motif VII(from amino acid residue 565 to residue 576 of SEQ ID NO: 2). SeeGoldstein and Weller, 1998, Virology, 244(2):442-57, the disclosure ofwhich is incorporated herein in its entirety for all purposes. Motif IIis one of the most highly conserved regions. Within this motif, theC-terminal 5 amino acids (336-GASLD-340) represent the most conservedcluster. Asp340 is an absolutely conserved amino acid, and aspartic acidresidues are required for metal binding in some endo- and exonucleases(Kovall and Matthews, 1997, Science, 277:1824-7). Within Motif II,Gly336 and Ser338 are absolutely conserved among 16 herpesvirushomologs. Goldstein and Weller demonstrated that the D340E mutant andG336A/S338A mutant of UL-12 lack exonuclease activity and therefore lackin vivo function.

In some embodiments, the exonuclease has a sequence that is at leastabout any of 70%, 80%, 90%, 95%, 98%, or 99% homologous to SEQ ID NO: 1.In some embodiments, the exonuclease comprises at least 1 (such as anyof 2, 3, 4, 5, 6, or 7) conserved motif of UL12.

In some embodiments, the exonuclease is of eukaryotic or viral origin.In some aspects, the exonuclease is EXOI (eukaryotic) or exo (phage). Inother embodiments, exonucleases such as ExoII or bacteriophage T7 gene 6exonuclease are used. In some embodiments, the exonuclease is Mre11,MRE11A, or MRE11B, for example of human origin.

Concomitant with, or sequential to, introduction of the exonucleaseand/or donor construct, a sequence-specific nuclease is introduced intothe cell. The term “sequence-specific endonuclease” or“sequence-specific nuclease,” as used herein, refers to a protein thatrecognizes and binds to a polynucleotide at a specific nucleic acidsequence and catalyzes a single- or double-strand break in thepolynucleotide. In certain embodiments, the sequence-specific nucleasecleaves the chromosome only once, i.e., a single double-strand break isintroduced at the insertion site during the methods described herein.

Examples of sequence-specific nucleases include zinc finger nucleases(ZFNs). ZFNs are recombinant proteins composed of DNA-binding zincfinger protein domains and effector nuclease domains. Zinc fingerprotein domains are ubiquitous protein domains, e.g., associated withtranscription factors, that recognize and bind to specific DNAsequences. One of the “finger” domains can be composed of about thirtyamino acids that include invariant histidine residues in complex withzinc. While over 10,000 zinc finger sequences have been identified thusfar, the repertoire of zinc finger proteins has been further expanded bytargeted amino acid substitutions in the zinc finger domains to createnew zinc finger proteins designed to recognize a specific nucleotidesequence of interest. For example, phage display libraries have beenused to screen zinc finger combinatorial libraries for desired sequencespecificity (Rebar et al., Science 263:671-673 (1994); Jameson et al,Biochemistry 33:5689-5695 (1994); Choo et al., PNAS 91: 11163-11167(1994), each of which is incorporated herein as if set forth in itsentirety). Zinc finger proteins with the desired sequence specificitycan then be linked to an effector nuclease domain, e.g., as described inU.S. Pat. No. 6,824,978, such as FokI, described in PCT ApplicationPublication Nos. WO1995/09233 and WO1994018313, each of which isincorporated herein by reference as if set forth in its entirety.

Another example of sequence-specific nucleases includes transcriptionactivator-like effector endonucleases (TALEN), which comprise a TALeffector domain that binds to a specific nucleotide sequence and anendonuclease domain that catalyzes a double-strand break at the targetsite. Examples of TALENs and methods of making and using are describedby PCT Patent Application Publication Nos. WO2011072246 and WO2013163628, and U.S. Application Publication No. US 20140073015 A1,incorporated herein by reference as if set forth in their entireties.

In one aspect, a transcription activator-like effector (TALE) modulateshost gene functions by binding specific sequences within gene promoters.“Transcription activator-like effector nucleases” or “TALENs” as usedinterchangeably herein refers to engineered fusion proteins of thecatalytic domain of a nuclease, such as endonuclease FokI, and adesigned TALE DNA-binding domain that may be targeted to a custom DNAsequence. A “TALEN monomer” refers to an engineered fusion protein witha catalytic nuclease domain and a designed TALE DNA-binding domain. TwoTALEN monomers may be designed to target and cleave a target region. Ingeneral, TALEs include tandem-like and nearly identical monomers (i.e.,repeat domains), flanked by N-terminal and C-terminal sequences. In someembodiments, each monomer contains 34 amino acids, and the sequence ofeach monomer is highly conserved. Only two amino acids per repeat (i.e.,residues 12th and 13th) are hypervariable, and are also known as repeatvariable di-residues (RVDs). The RVDs determine the nucleotide-bindingspecificity of each TALE repeat domain. RVDs or RVD modules typicallyinclude 33-35 amino acids, of the TALE DNA-binding domain. RVD modulesmay be combined to produce an RVD array. The “RVD array length” as usedherein refers to the number of RVD modules that corresponds to thelength of the nucleotide sequence within the target region that isrecognized by the TALEN, i.e., the binding region.

TALENs may be used to introduce site-specific double-strand breaks attargeted genomic loci. Site-specific double-strand breaks are createdwhen two independent TALENs bind to nearby DNA sequences, therebypermitting dimerization of FokI and cleavage of the target DNA. TALENshave advanced genome editing due to their high rate of successful andefficient genetic modification. This DNA cleavage may stimulate thenatural DNA-repair machinery, leading to one of two possible repairpathways: homology-directed repair (HDR) or the non-homologous endjoining (NHEJ) pathway. The TALENs may be designed to target any gene,including genes involved in a genetic disease. The TALENs may include anuclease and a TALE DNA-binding domain that binds to the target gene.The target gene may have a mutation such as a frameshift mutation or anonsense mutation. If the target gene has a mutation that causes apremature stop codon, the TALEN may be designed to recognize and bind anucleotide sequence upstream or downstream from the premature stopcodon. In some embodiments, the TALE DNA-binding domain may have an RVDarray length between 1-30 modules, between 1-25 modules, between 1-20modules, between 1-15 modules, between 5-30 modules, between 5-25modules, between 5-20 modules, between 5-15 modules, between 7-25modules, between 7-23 modules, between 7-20 modules, between 10-30modules, between 10-25 modules, between 10-20 modules, between 10-15modules, between 15-30 modules, between 15-25 modules, between 15-20modules, between 15-19 modules, between 16-26 modules, between 16-41modules, between 20-30 modules, or between 20-25 modules in length. TheRVD array length may be about any of 5 modules, 8 modules, 10 modules,11 modules, 12 modules, 13 modules, 14 modules, 15 modules, 16 modules,17 modules, 18 modules, 19 modules, 20 modules, 22 modules, 25 modulesor 30 modules.

Another example of a sequence-specific nuclease system that can be usedwith the methods and compositions described herein includes theCas/CRISPR system (Wiedenheft, B. et al. Nature 482, 331-338 (2012);Jinek, M. et al. Science 337, 816-821 (2012); Mali, P. et al. Science339, 823-826 (2013); Cong, L. et al. Science 339, 819-823 (2013)). TheCas/CRISPR (Clustered Regularly interspaced Short Palindromic Repeats)system exploits RNA-guided DNA-binding and sequence-specific cleavage oftarget DNA. A guide RNA (gRNA) contains about 20-25 (such as 20)nucleotides that are complementary to a target genomic DNA sequenceupstream of a genomic PAM (protospacer adjacent motifs) site and aconstant RNA scaffold region. In certain embodiments, the targetsequence is associated with a PAM, which is a short sequence recognizedby the CRISPR complex. The precise sequence and length requirements forthe PAM differ depending on the CRISPR enzyme used, but PAMs aretypically 2-5 bp sequences adjacent to the protospacer (that is, thetarget sequence). Examples of PAM sequences are known in the art, andthe skilled person will be able to identify further PAM sequences foruse with a given CRISPR enzyme. For example, target sites for Cas9 fromS. pyogenes, with PAM sequences NGG, may be identified by searching for5′-N_(x)-NGG-3′ both on an input sequence and on the reverse-complementof the input. In certain embodiments, the genomic PAM site used hereinis NGG, NNG, NAG, NGGNG, or NNAGAAW. Other PAM sequences and methods foridentifying PAM sequences are known in the art, for example, asdisclosed in U.S. Pat. No. 8,697,359, the disclosure of which isincorporated herein by reference for all purposes. In particularembodiments, the Streptococcus pyogenes Cas9 (SpCas9) is used and thecorresponding PAM is NGG. In some aspects, different Cas9 enzymes fromdifferent bacterial strains use different PAM sequences. The Cas(CRISPR-associated) protein binds to the gRNA and the target DNA towhich the gRNA binds and introduces a double-strand break in a definedlocation upstream of the PAM site. In one aspect, the CRISPR/Cas,Cas/CRISPR, or the CRISPR-Cas system (these terms are usedinterchangeably throughout this application) does not require thegeneration of customized proteins to target specific sequences butrather a single Cas enzyme can be programmed by a short RNA molecule torecognize a specific DNA target, i.e., the Cas enzyme can be recruitedto a specific DNA target using the short RNA molecule.

In some embodiments, the sequence-specific nuclease is a type II Casprotein. In some embodiments, the sequence-specific nuclease is Cas9(also known as Csn1 and Csx12), a homolog thereof, or a modified versionthereof. In some embodiments, a combination of two or more Cas proteinscan be used. In some embodiments the CRISPR enzyme is Cas9, and may beCas9 from S. pyogenes or S. pneumoniae. The Cas enzymes are known in theart; for example, the amino acid sequence of S. pyogenes Cas9 proteinmay be found in the SwissProt database under accession number Q99ZW2.

In some embodiments, Cas9 is used in the methods described herein. Cas9harbors two independent nuclease domains homologous to HNH and RuvCendonucleases, and by mutating either of the two domains, the Cas9protein can be converted to a nickase that introduces single-strandbreaks (Cong, L. et al. Science 339, 819-823 (2013)). It is specificallycontemplated that the inventive methods and compositions can be usedwith the single- or double-strand-inducing version of Cas9, as well aswith other RNA-guided DNA nucleases, such as other bacterial Cas9-likesystems. The sequence-specific nuclease of the methods and compositionsdescribed herein can be engineered, chimeric, or isolated from anorganism.

CRISPRs, also known as SPIDRs (SPacer Interspersed Direct Repeats),constitute a family of DNA loci that are usually specific to aparticular bacterial species. The CRISPR locus comprises a distinctclass of interspersed short sequence repeats (SSRs) that are recognizedin E. coli (Ishino et al., 1987, J. Bacteriol., 169:5429-5433; andNakata et al., 1989, J. Bacteriol., 171:3553-3556), and associatedgenes. Similar interspersed SSRs have been identified in Haloferaxmediterranei, Streptococcus pyogenes, Anabaena, and Mycobacteriumtuberculosis (See, Groenen et al., 1993, Mol. Microbiol., 10:1057-1065;Hoe et al., 1999, Emerg, Infect. Dis., 5:254-263; Masepohl et al., 1996,Biochim, Biophys, Acta 1307:26-30; and Mojica et at, 1995, MolMicrobiol., 17:85-93). The CRISPR loci typically differ from other SSRsby the structure of the repeats, which have been termed short regularlyspaced repeats (SRSRs) (Janssen et al., 2002, OMICS J. Integ. Biol.,6:23-33; and Mojica et al., 2000, Mol. Microbiol, 36:244-246). Ingeneral, the repeats are short elements that occur in clusters that areregularly spaced by unique intervening sequences with a substantiallyconstant length (Mojica et al., 2000, supra). Although the repeatsequences are highly conserved between strains, the number ofinterspersed repeats and the sequences of the spacer regions typicallydiffer from strain to strain (van Embden et al., 2000, J. Bacteriol.,182:2393-2401). CRISPR loci have been identified in more than 40prokaryotes (See e.g., Jansen et al., 2002, Mol. Microbiol.,43:1565-1575) including, but not limited to Aeropyrum, Pyrobaculum,Sulfolobus, Archaeoglobus, Halocarcula, Methanobacteriumn,Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus,Thernioplasnia, Corynebacterium, Mycobacterium, Streptomyces, Aquifrx,Porphvromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus,Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus,Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter,Myrococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia,Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium,Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.

The CRISPR system refers collectively to transcripts and other elementsinvolved in the expression of or directing the activity of Cas genes,including sequences encoding a Cas gene, a tracr (trans-activatingCRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), atracr-mate sequence (encompassing a “direct repeat” and atracrRNA-processed partial direct repeat in the context of an endogenousCRISPR system), a guide sequence (also referred to as a “spacer” in thecontext of an endogenous CRISPR system), or other sequences andtranscripts from a CRISPR locus. In some embodiments, one or moreelements of a CRISPR system is derived from a type I, type II, or typeIII CRISPR system. In some embodiments, one or more elements of a CRISPRsystem can be derived from a particular organism comprising anendogenous CRISPR system, such as Streptococcus pyogenes. In certainembodiments, elements of a CRISPR system promote the formation of aCRISPR complex at the site of a target sequence (also referred to as aprotospacer in the context of an endogenous CRISPR system). In thecontext of formation of a CRISPR complex, “target sequence” refers to asequence to which a guide sequence is designed to have complementarity,where hybridization between a target sequence and a guide sequencepromotes the formation of a CRISPR complex. Full complementarity is notnecessarily required, provided there is sufficient complementarity tocause hybridization and promote formation of a CRISPR complex, A targetsequence may comprise any polynucleotide, such as DNA or RNApolynucleotides. In some embodiments, a target sequence is located inthe nucleus or cytoplasm of a cell. In some embodiments, the targetsequence may be within an organelle of a eukaryotic cell, for example,mitochondrion or chloroplast. A sequence or template that may be usedfor recombination into the targeted locus comprising the targetsequences is referred to as an “editing template” or “editingpolynucleotide” or “editing sequence”. In aspects of the presentdisclosure, an exogenous template polynucleotide may be referred to asan editing template. In some aspects, the recombination is homologousrecombination. The CRISPR-Cas systems have been used for editing,regulating and targeting genomes, for example, as disclosed in Sanderand Joung, 2014 Nature Biotechnology 32(4): 347-55, the disclosure ofwhich is incorporated herein by reference for all purposes.

An exemplary type II CRISPR system is the type II CRISPR locus fromStreptococcus pyogenes SF370, which contains a cluster of four genesCas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements,tracrRNA and a characteristic array of repetitive sequences (directrepeats) interspaced by short stretches of non-repetitive sequences(spacers, about 30 bp each). In this system, targeted DNA double-strandbreak (DSB) is generated in four sequential steps. First, two non-codingRNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPRlocus. Second, tracrRNA hybridizes to the direct repeats of pre-crRNA,which is then processed into mature crRNAs containing individual spacersequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to theDNA target consisting of the protospacer and the corresponding PAM viaheteroduplex formation between the spacer region of the crRNA and theprotospacer DNA. Finally, Cas9 mediates cleavage of target DNA upstreamof PAM to create a DSB within the protospacer. Additional descriptionsof CRISPR and/or Cas and methods of use can be found in WO 2007025097,US 20100093617, US 20130011828, U.S. Ser. No. 13/960,796, U.S. Pat. No.8,546,553, WO 2010011961, US 20140093941, US 20100076057, US20110217739, WO 2010075424, WO 2013142578, WO 2013141680, US20130326645, WO 2013169802, US 20140068797, WO 2013176772, WO2013181440, US 20130330778, WO 2013188037, WO 2013188522, WO 2013188638,WO 2013192278, WO 2014018423, CN 103388006, WO 2014022702, US20140090113, WO 2014039872, WO 2014065596, U.S. Pat. No. 8,697,359, andCN 103725710, the disclosures of which are incorporated herein byreference in their entireties for all purposes.

In some embodiments, the sequence-specific nuclease is an RNA-guidedendonuclease (for example for the Cas/CRIPSR) system. The term“RNA-guided DNA nuclease” or “RNA-guided DNA nuclease” or “RNA-guidedendonuclease,” as used herein, refers to a protein that recognizes andbinds to a guide RNA and a polynucleotide, e.g., a target gene, at aspecific nucleotide sequence and catalyzes a single- or double-strandbreak in the polynucleotide. In some embodiments, a guide RNA is an RNAcomprising a 5′ region comprising at least one repeat from a CRISPRlocus and a 3′ region that is complementary to the predeterminedinsertion site on the chromosome. In certain embodiments, the 5′ regioncomprises a sequence that is complementary to the predeterminedinsertion site on the chromosome, and the 3′ region comprises at leastone repeat from a CRISPR locus. In some aspects, the 3′ region of theguide RNA further comprises the one or more structural sequences ofcrRNA and/or trRNA. The 5′ region can comprise, for example, about 1, 2,3, 4, 5, or more repeats from a CRISPR locus, and can be about any of 5,10, 15, 20, 25, 30, or more nucleotides long. In some embodiments, the5′ region sequence that is complementary to the predetermined insertionsite on the chromosome comprises between about 17 and about 24nucleotides. In other embodiments, the 3′ region can be, for example,about any of 5, 10, 15, 20, 25, 30, or more nucleotides long. In someaspects, 5′ region sequence that is complementary to the predeterminedinsertion site on the chromosome can vary in length, while the 3′ regionsequence is fixed in length. In such embodiments where guide RNA isneeded, the introduction of the sequence-specific nuclease step may alsocomprise introduction of the guide RNA into the cell. The guide RNA canbe introduced, for example, as RNA or as a plasmid or other nucleic acidvector encoding the guide RNA. The plasmid or other nucleic acid vectormay further comprise coding sequence(s) for the sequence-specificnuclease (such as Cas) and/or the exonuclease (such as UL-12). In someembodiments, the guide RNA comprises a crRNA and a tracrRNA, and the twopieces of RNA form a complex through hybridization. In some embodimentswhen multiple guide RNAs are used, a single tracrRNA paired withdifferent crRNAs can be used.

Thus, for example, in some embodiments, there is provided a method ofinserting a donor sequence at a predetermined insertion site on achromosome in an eukaryotic cell, the method comprising: a) introducinginto the cell a sequence-specific RNA-guided nuclease (such as Cas, forexample Cas9); b) introducing into the cell a guide RNA recognizing theinsertion site; c) introducing into the cell a donor construct; and d)introducing into the cell a nucleic acid sequence encoding anexonuclease (such as UL-12); wherein the donor construct is a linearnucleic acid or cleaved within the cell to produce a linear nucleicacid, wherein the linear nucleic acid comprises a 5′ homology arm, thedonor sequence, and a 3′ homology arm, wherein the 5′ homology arm ishomologous to a sequence upstream of the nuclease cleavage site on thechromosome and wherein the 3′ homology arm is homologous to a sequencedownstream of the nuclease cleavage site on the chromosome; wherein the5′ homology arm and the 3′ homology arm are proximal to the 5′ and 3′ends of the linear nucleic acid, respectively, and wherein the donorsequence is inserted into the chromosome at the insertion site throughhomologous recombination.

In some embodiments, there is provided a method of inserting a donorsequence at a predetermined insertion site on a chromosome in aneukaryotic cell, the method comprising: a) introducing into the cell anucleic acid sequence encoding a sequence-specific RNA-guided nuclease(such as Cas, for example Cas9); b) introducing into the cell a nucleicacid sequence encoding a guide RNA recognizing the insertion site; c)introducing into the cell a donor construct; and d) introducing into thecell a nucleic acid sequence encoding an exonuclease (such as UL-12);wherein the donor construct is a linear nucleic acid or cleaved withinthe cell to produce a linear nucleic acid, wherein the linear nucleicacid comprises a 5′ homology arm, the donor sequence, and a 3′ homologyarm, wherein the 5′ homology arm is homologous to a sequence upstream ofthe nuclease cleavage site on the chromosome and wherein the 3′ homologyarm is homologous to a sequence downstream of the nuclease cleavage siteon the chromosome; wherein the 5′ homology arm and the 3′ homology armare proximal to the 5′ and 3′ ends of the linear nucleic acid,respectively, and wherein the donor sequence is inserted into thechromosome at the insertion site through homologous recombination.

In some embodiments, there is provided a method of inserting a donorsequence at a predetermined insertion site on a chromosome in aneukaryotic cell, the method comprising: a) introducing into the cell avector comprising a nucleic acid sequence encoding a sequence-specificRNA-guided nuclease (such as Cas, for example Cas9); b) introducing intothe cell a vector comprising a nucleic acid sequence encoding a guideRNA recognizing the insertion site; c) introducing into the cell a donorconstruct; and d) introducing into the cell a DNA vector comprising anucleic acid sequence encoding an exonuclease (such as UL-12); whereinthe donor construct is a linear nucleic acid or cleaved within the cellto produce a linear nucleic acid, wherein the linear nucleic acidcomprises a 5′ homology arm, the donor sequence, and a 3′ homology arm,wherein the 5′ homology arm is homologous to a sequence upstream of thenuclease cleavage site on the chromosome and wherein the 3′ homology armis homologous to a sequence downstream of the nuclease cleavage site onthe chromosome; wherein the 5′ homology arm and the 3′ homology arm areproximal to the 5′ and 3′ ends of the linear nucleic acid, respectively,and wherein the donor sequence is inserted into the chromosome at theinsertion site through homologous recombination.

In some embodiments, there is provided a method of inserting a donorsequence at a predetermined insertion site on a chromosome in aneukaryotic cell, the method comprising: a) introducing into the cell avector comprising a nucleic acid sequence encoding a sequence-specificRNA-guided nuclease (such as Cas, for example Cas9) and a guide RNArecognizing the insertion site; b) introducing into the cell a donorconstruct; and c) introducing into the cell a DNA vector comprising anucleic acid sequence encoding an exonuclease (such as UL-12); whereinthe donor construct is a linear nucleic acid or cleaved within the cellto produce a linear nucleic acid, wherein the linear nucleic acidcomprises a 5′ homology arm, the donor sequence, and a 3′ homology arm,wherein the 5′ homology arm is homologous to a sequence upstream of thenuclease cleavage site on the chromosome and wherein the 3′ homology armis homologous to a sequence downstream of the nuclease cleavage site onthe chromosome; wherein the 5′ homology arm and the 3′ homology arm areproximal to the 5′ and 3′ ends of the linear nucleic acid, respectively,and wherein the donor sequence is inserted into the chromosome at theinsertion site through homologous recombination.

In some embodiments, there is provided a method of inserting a donorsequence at a predetermined insertion site on a chromosome in aneukaryotic cell, the method comprising: a) introducing into the cell avector comprising a nucleic acid sequence encoding a sequence-specificRNA-guided nuclease (such as Cas, for example Cas9) and a nucleic acidsequence encoding an exonuclease (such as UL-12); b) introducing intothe cell a guide RNA recognizing the insertion site; and c) introducinginto the cell a donor construct; wherein the donor construct is a linearnucleic acid or cleaved within the cell to produce a linear nucleicacid, wherein the linear nucleic acid comprises a 5′ homology arm, thedonor sequence, and a 3′ homology arm, wherein the 5′ homology arm ishomologous to a sequence upstream of the nuclease cleavage site on thechromosome and wherein the 3′ homology arm is homologous to a sequencedownstream of the nuclease cleavage site on the chromosome; wherein the5′ homology arm and the 3′ homology arm are proximal to the 5′ and 3′ends of the linear nucleic acid, respectively, and wherein the donorsequence is inserted into the chromosome at the insertion site throughhomologous recombination.

In some embodiments, there is provided a method of inserting a donorsequence at a predetermined insertion site on a chromosome in aneukaryotic cell, the method comprising: a) introducing into the cell avector comprising a nucleic acid sequence encoding a sequence-specificnuclease (such as Cas, for example Cas9), a guide RNA recognizing theinsertion site, and a nucleic acid sequence encoding an exonuclease(such as UL-12); b) introducing into the cell a donor construct; whereinthe donor construct is a linear nucleic acid or cleaved within the cellto produce a linear nucleic acid, wherein the linear nucleic acidcomprises a 5′ homology arm, the donor sequence, and a 3′ homology arm,wherein the 5′ homology arm is homologous to a sequence upstream of thenuclease cleavage site on the chromosome and wherein the 3′ homology armis homologous to a sequence downstream of the nuclease cleavage site onthe chromosome; wherein the 5′ homology arm and the 3′ homology arm areproximal to the 5′ and 3′ ends of the linear nucleic acid, respectively,and wherein the donor sequence is inserted into the chromosome at theinsertion site through homologous recombination.

In some embodiments, there is provided a method of inserting a donorsequence at a predetermined insertion site on a chromosome in aneukaryotic cell (such as a zygotic cell), the method comprising: a)injecting into the cell an mRNA sequence encoding a sequence-specificnuclease (such as Cas, for example Cas9); b) injecting into the cell aguide RNA recognizing the insertion site; c) introducing (such asinjecting) into the cell a donor construct; and d) injecting into thecell an mRNA sequence encoding an exonuclease; wherein the donorconstruct is a linear nucleic acid or cleaved within the cell to producea linear nucleic acid, wherein the linear nucleic acid comprises a 5′homology arm, the donor sequence, and a 3′ homology arm, wherein the 5′homology arm is homologous to a sequence upstream of the nucleasecleavage site on the chromosome and wherein the 3′ homology arm ishomologous to a sequence downstream of the nuclease cleavage site on thechromosome; wherein the 5′ homology arm and the 3′ homology arm areproximal to the 5′ and 3′ ends of the linear nucleic acid, respectively,and wherein the donor sequence is inserted into the chromosome at theinsertion site through homologous recombination. In some embodiments,the injection is carried out in vitro. In some embodiments, theinjection is carried out in vivo. In some embodiments, the methodfurther comprises transcribing in vitro a nucleic acid encoding thesequence-specific RNA-guided nuclease into mRNA. In some embodiments,the method further comprises transcribing in vitro a nucleic acidencoding the guide RNA recognizing the insertion site into mRNA. In someembodiments, the method further comprises transcribing in vitro anucleic acid encoding the exonuclease into mRNA.

The insertion of the donor sequence can be evaluated using any methodsknown in the art. For example, a 5′ primer corresponding to a sequenceupstream of the 5′ homology arm and a corresponding 3′ primercorresponding to a region in the donor sequence can be designed toassess the 5′-junction of the insertion. Similarly, a 3′ primercorresponding to a sequence downstream of the 3′ homology arm and acorresponding 5′ primer corresponding to a region in the donor sequencecan be designed to assess the 3′-junction of the insertion. Othermethods such as southern blot and DNA sequencing technologies can alsobe used.

The insertion site can be at any desired site, so long assequence-specific nuclease can be designed to effect cleavage at suchsite. In some embodiments, the insertion site is at a target gene locus.In some embodiments, the insertion site is not a gene locus.

“Donor sequence” as used herein refers to a nucleic acid to be insertedinto the chromosome of a host cell. In some embodiments, the donornucleic acid is a sequence not present in the host cell. In someembodiments, the donor sequence is an endogenous sequence present at asite other than the predetermined target site. In some embodiments, thedonor sequence is a coding sequence. In some embodiments, the donorsequence is a non-coding sequence. In some embodiments, the donorsequence is a mutant locus of a gene.

The size of the donor sequence can range from about 1 bp to about 100kb. In certain embodiments, the size of the donor sequence is betweenabout 1 bp and about 10 bp, between about 10 bp and about 50 bp, betweenabout 50 bp and about 100 bp, between about 100 bp and about 500 bp,between about 500 bp and about 1 kb, between about 1 kb and about 10 kb,between about 10 kb and about 50 kb, between about 50 kb and about 100kb, or more than about 100 kb.

In some embodiments, the donor sequence is an exogenous gene to beinserted into the chromosome. In some embodiments, the donor sequence ismodified sequence that replaces the endogenous sequence at the targetsite. For example, the donor sequence may be a gene harboring a desiredmutation, and can be used to replace the endogenous gene present on thechromosome. In some embodiments, the donor sequence is a regulatoryelement. In some embodiments, the donor sequence is a tag or a codingsequence encoding a reporter protein and/or RNA. In some embodiments,the donor sequence is inserted in frame into the coding sequence of atarget gene which will allow expression of a fusion protein comprisingan exogenous sequence fused to the N- or C-terminus of the targetprotein.

The donor construct described herein is either a linear nucleic acid orcleaved within the cell to produce a linear nucleic acid. The linearnucleic acid described herein comprises a 5′ homology arm, a donorsequence, and a 3′ homology arm. The 5′ and 3′ homology arms arehomologous to a sequence upstream and downstream of the DNA cleavagesite on the target chromosome, thereby allowing homologous recombinationto occur.

The term “homology” or “homologous” as used herein is defined as thepercentage of nucleotide residues in the homology arm that are identicalto the nucleotide residues in the corresponding sequence on the targetchromosome, after aligning the sequences and introducing gaps, ifnecessary, to achieve the maximum percent sequence identity. Alignmentfor purposes of determining percent nucleotide sequence homology can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN, ClustalW2 or Megalign (DNASTAR) software. Those skilledin the art can determine appropriate parameters for aligning sequences,including any algorithms needed to achieve maximal alignment over thefull length of the sequences being compared. In some embodiments, thehomology between the 5′ homology arm and the corresponding sequence onthe chromosome is at least about any of 80%, 85%, 90%, 95%, 98%, 99%, or100%. In some embodiments, the homology between the 3′ homology arm andthe corresponding sequence on the chromosome is at least about any of80%, 85%, 90%, 95%, 98%, 99%, or 100%.

In one embodiment, the homology arms are more than about 30 bp inlength, for example more than about any of 50 bp, 100 bp, 200 bp, 300bp, 500 bp, 800 bp, 1 kb, 1.5 kb, 2 kb and 5 kb in length. The 5′ and/or3′ homology arms can be homologous to a sequence immediately upstreamand/or downstream of the DNA cleavage site. Alternatively, the 5′ and/or3′ homology arms can be homologous to a sequence that is distant fromthe DNA cleavage site, for example a sequence that is 0 bp away from theDNA cleavage site, or partially or completely overlaps with the DNAcleavage site. In other embodiments, the 5′ and/or 3′ homology arms canbe homologous to a sequence that is at least about 1, 2, 5, 10, 15, 20,25, 30, 50, 100, 200, 300, 400, or 500 bp away from the DNA cleavagesite.

The 5′ and 3′ homology arms of the linear nucleic acid are each proximalto the 5′ and 3′ ends of the linear nucleic acid, respectively, i.e., nomore than about 200 bp away from the 5′ and 3′ ends of the linearnucleic acid. In some embodiments, the 5′ homology arm is no more thanabout any of 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70,80, 90, 100, 120, 140, 160, 180, or 200 bp away from the 5′ end of thelinear DNA. In some embodiments, the 3′ homology arm is no more thanabout any of 1, 2, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70,80, 90, 100, 120, 140, 160, 180, or 200 bp away from the 3′ end of thelinear DNA. In some aspects, the 5′ and/or 3′ homology arms can beimmediately linked to the 5′ and 3′ ends of the linear DNA,respectively, or partially or completely overlap with the 5′ and 3′ endsof the linear DNA, respectively.

In some embodiments, the donor construct is cleaved within the cell (forexample by a sequence-specific nuclease recognizing a cleavage site onthe construct) to produce a linear nucleic acid described herein. Forexample, the donor construct may comprise flanking sequences upstream ofthe 5′ homology arm and downstream of the 3′ homology arm. Such flankingsequences in some embodiments do not exist in the genomic sequences ofthe host cell thus allowing cleavage to only occur on the donorconstruct. Sequence-specific nucleases can then be designed accordinglyto effect cleavage at the flanking sequences that allows the release ofthe linear nucleic acid without affecting the host sequences. Theflanking sequences can be, for example, about 5 to about 500 bp,including about any of 5-15, 15-30, 30-50, 50-80, 80-100, 100-150,150-200, 200-300, 300-400, or 400-500 bp. In some embodiments, theflanking sequence is no more than about any of 10, 20, 30, 40, 50, 60,70, 80, 90, or 100 bp. In some embodiments, the portion of the flankingsequence remaining on the linear nucleic acid after sequence-specificcleavage is about any of 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 bp.In some embodiments, the flanking sequence comprises any one of thefollowing sequences:

(SEQ ID NO: 3) GGCAGAAATGGCTCCGATCGAGG (SEQ ID NO: 4)GGGCGGGATTGATAGCGCGCGGG (SEQ ID NO: 5) GGCAGTCGGGAACATCTCGTGGG(SEQ ID NO: 6) GGGCGCAGTAATTCTTAGAGCGG (SEQ ID NO: 7)GGCTAATAACTTAATCGTGGAGG (SEQ ID NO: 8) GGTTAAGCCTTATTGGTGGTCGG(SEQ ID NO: 9) GGAGGCCTGCTTGCAAGCATTGG (SEQ ID NO: 10)GGTTAGGCCCTAAGCGAATACGG (SEQ ID NO: 11) GGAGCCGAGTTGACGGTTAGCGG(SEQ ID NO: 12) GGGGTTCCTTCACGAGCGTCCGG (SEQ ID NO: 13)GGTACAATGTAACGTTGCGCGGG (SEQ ID NO: 14) GGTATTCAAGTCACTAATGTCGG(SEQ ID NO: 15) GGAACCCCTTCCGTTCCGTCGGG (SEQ ID NO: 16)GGTATTCACTCCTAAAGCGTCGG (SEQ ID NO: 17) GGGATGGAACACTAGACTGCGGG(SEQ ID NO: 18) GGTTAATCCCTCATGACCGTCGG (SEQ ID NO: 19)GGAGCTTCAGTGTCGGTCGTTGG (SEQ ID NO: 20) GGTTACGTGCCATATACGTTCGG

In some embodiments, the donor construct is a circular DNA construct.The donor construct can further include certain sequences that providestructural or functional support, such as sequences of a plasmid orother vector that supports propagation of the donor construct (e.g.,pUC19 vector). The donor construct can optionally also include certainselectable markers or reporters, some of which may be flanked byrecombinase recognition sites for subsequent activation, inactivation,or deletion.

In some embodiments when the donor construct is cleaved within the cellto produce a linear nucleic acid, the methods described herein mayfurther comprise introducing into the cell a second sequence-specificnuclease into the cell. The second sequence-specific nuclease recognizescleavage sites (such as flanking sequences described herein) on theconstruct and lead to cleavage of the donor construct within the cell,producing a linear nucleic acid.

Thus, in some embodiments there is provided a method of inserting adonor sequence at a predetermined insertion site on a chromosome of aneukaryotic cell, comprising: a) introducing into the cell a firstsequence-specific nuclease that cleaves the chromosome at the insertionsite; b) introducing into the cell a donor construct (such as a circulardonor construct), c) introducing into the cell a secondsequence-specific nuclease that cleaves the donor construct; and d)introducing into the cell an exonuclease; wherein upon cleavage by thesecond sequence-specific nuclease the donor construct produces a linearnucleic acid comprising a 5′ homology arm, the donor sequence, and a 3′homology arm, wherein the 5′ homology arm is homologous to a sequenceupstream of the nuclease cleavage site on the chromosome and wherein the3′ homology arm is homologous to a sequence downstream of the nucleasecleavage site on the chromosome; and wherein the 5′ homology arm and the3′ homology arm are proximal to the 5′ and 3′ ends of the linear nucleicacid, respectively, and wherein the donor sequence is inserted into thechromosome at the insertion site through homologous recombination. Insome embodiments, the first sequence-specific nuclease and the secondsequence-specific nuclease are of the same kind (for example, both areZFN, TALEN, or CRISPR-based nucleases). In some embodiments, the firstsequence-specific nuclease and the second sequence-specific nuclease areof different kinds. In some embodiments, the first sequence-specificnuclease, the second sequence-specific nuclease, and/or the exonucleaseare introduced into the cell simultaneously. In some embodiments, thefirst sequence-specific nuclease, the second sequence-specific nuclease,and/or the exonuclease are introduced into the cell sequentially. Insome embodiments, the first sequence-specific nuclease, the secondsequence-specific nuclease, and/or the exonuclease are introduced intothe cell as a cDNA. In some embodiments, the first sequence-specificnuclease, the second sequence-specific nuclease, and/or the exonucleaseare introduced into the cell as an mRNA. In some embodiments, the firstsequence-specific nuclease, the second sequence-specific nuclease,and/or the exonuclease are introduced into the cell as a protein.

In some embodiments, the first sequence-specific nuclease and the secondsequence-specific nuclease are both sequence-specific RNA-guidednucleases. In such embodiments, a single nuclease together with twodifferent guide RNAs (one recognizing the insertion site, anotherrecognizing the cleavage site on the donor construct) can be used. Forexample, in some embodiments, the method comprises: a) introducing intothe cell a sequence-specific RNA-guided nuclease; b) introducing intothe cell a donor construct (such as a circular donor construct); c)introducing into the cell a first guide RNA recognizing the insertionsite; d) introducing into the cell a second guide RNA recognizing acleavage site on the donor construct; and e) introducing into the cellan exonuclease; wherein upon cleavage by the sequence-specific nucleasethe donor construct produces a linear nucleic acid comprising a 5′homology arm, the donor sequence, and a 3′ homology arm, wherein the 5′homology arm is homologous to a sequence upstream of the nucleasecleavage site on the chromosome and wherein the 3′ homology arm ishomologous to a sequence downstream of the nuclease cleavage site on thechromosome; and wherein the 5′ homology arm and the 3′ homology arm areproximal to the 5′ and 3′ ends of the linear nucleic acid, respectively,and wherein the donor sequence is inserted into the chromosome at theinsertion site through homologous recombination. In some embodiments,the first guide RNA and the second guide RNA are introduced into thecell via a DNA vector (and in some embodiments on the same vector). Insome embodiments, the first guide RNA and the second guide RNA areintroduced into the cells by injection (for example after being producedby in vitro transcription).

The present application therefore also provides methods of producing alinear nucleic acid described herein. In some embodiments, there isprovided a method of producing a linear nucleic acid in an eukaryoticcell, wherein the linear nucleic acid comprises a 5′ homology arm, thedonor sequence, and a 3′ homology arm, wherein the 5′ homology arm ishomologous to a sequence upstream of the nuclease cleavage site on thechromosome and wherein the 3′ homology arm is homologous to a sequencedownstream of the nuclease cleavage site on the chromosome; and whereinthe 5′ homology arm and the 3′ homology arm are proximal to the 5′ and3′ ends of the linear nucleic acid, respectively, comprising: a)introducing into the cell a circulated donor construct comprising thelinear nucleic acid and further comprising a 5′ flanking sequenceupstream of the 5′ homology arm and a 3′ flanking sequence downstream ofthe 3′ homology arm; and b) introducing into the cell asequence-specific nuclease, wherein the sequence-specific nucleasecleaves the circular nucleic acid construct at the flanking sequencesthereby producing the linear nucleic acid. In some embodiments, thecleavage site is at the 5′ flanking sequence and/or 3′ flankingsequence. In some embodiments, the linear DNA is about 200 bp to about100 kb long. In certain embodiments, the linear DNA is between about 10bp and about 50 bp, between about 50 bp and about 100 bp, between about100 bp and about 150 bp, between about 150 bp and about 200 bp, betweenabout 200 bp and about 500 bp, between about 500 bp and about 1 kb,between about 1 kb and about 10 kb, between about 10 kb and about 50 kb,between about 50 kb and about 100 kb, or more than about 100 kb inlength.

Uses of the Present Methods

The methods described herein can find many uses. For example, themethods described herein can be useful for generating gene-modifiedcells (such as immune cells), which can be useful for cellulartherapeutics.

In some embodiments, there is provided a method of generating agenetically modified animal (for example a genetically modified rodentsuch as mouse or rat) comprising a donor sequence inserted at apredetermined insertion site on the chromosome of the animal,comprising: a) introducing into a cell of the animal a sequence-specificnuclease that cleaves the chromosome at the insertion site; b)introducing into the cell a donor construct; c) introducing into thecell an exonuclease; wherein the donor construct is a linear nucleicacid or cleaved within the cell to produce a linear nucleic acid; and d)introducing the cell into a carrier animal to produce the geneticallymodified animal, wherein the donor construct is a linear nucleic acid orcleaved within the cell to produce a linear nucleic acid, wherein thelinear nucleic acid comprises a 5′ homology arm, the donor sequence, anda 3′ homology arm, wherein the 5′ homology arm is homologous to asequence upstream of the nuclease cleavage site on the chromosome andwherein the 3′ homology arm is homologous to a sequence downstream ofthe nuclease cleavage site on the chromosome; wherein the 5′ homologyarm and the 3′ homology arm are proximal to the 5′ and 3′ ends of thelinear nucleic acid, respectively, and wherein the donor sequence isinserted into the chromosome at the insertion site through homologousrecombination. In some embodiments, the cell is an embryotic stem cell.In some embodiments, the cell is a zygotic cell. In some embodiments,the method further comprises breeding the genetically modified animal.

In some embodiments, the cells are cells from a blastocyst. Uponinjection of the various components into the cells, chimeric animals canbe developed from the injected blastocysts. The heterozygous F1 animalscan be obtained by breeding between the chimera and the pure inbredanimal. The homozygous animals can be obtained by inter-cross betweenthe heterozygous animals.

In some embodiments, the method is used to generate a mutant animalhaving a specific mutant allele. For example, the donor sequence maycontain a mutant allele, and can be inserted into the genome of theanimal (for example by replacing the corresponding endogenous locus).The mutant animals can be useful for many purposes, for example servingas a research tool or a disease model. In some embodiments, the animalis modified to have a desired phenotype, such as a desired diseasephenotype. Animals with various disease phenotypes that can be generatedby methods described herein include, but are not limited to, animalsexhibiting a phenotype in metabolic diseases, immunological diseases,neurological diseases, neurodegenerative diseases (such as Alzheimer'sdisease), embryonic development diseases, vascular diseases,inflammatory diseases (such as asthma and arthritis), infectiousdiseases, cancer, behavioral diseases, and cognitive diseases.

In some embodiments, the method described herein is used to generate a“humanized” animal, such as a humanized mouse or a humanized rat. A“humanized animal” used herein refers to an animal harboring a donorsequence of human origin. The human donor sequence can be inserted atany site on the genome. In some embodiments, the human donor sequence isinserted at the corresponding endogenous locus in the animal cell.

In some embodiments, a humanized rodent capable of producingimmunoglobulin comprising a human variable domain and/or human constantdomain can be generated. A rearranged or unrearranged humanimmunoglobulin locus containing the human immunoglobulin V, D, J and/orconstant loci can be placed on the donor construct described herein andintroduced into the genome of the animal cell by homologousrecombination. The human immunoglobulin in some embodiments is insertedat the corresponding endogenous immunoglobulin locus in the animal cell.Through such manipulation transgenic animals capable of producing fullyhuman antibodies, chimeric antibodies (e.g., antibodies comprising mousevariable domains and human constant domains), or reverse chimericantibodies (e.g., antibodies comprising human variable domains and mouseconstant regions), can be generated.

In some embodiments, the method described herein is used to generate amouse model for a human disease or condition. In certain aspects, themouse model reflects or mimics at least one aspect of the human diseaseor condition. In other aspects, the compositions and methods disclosedherein are used to knock-in a human gene into the mouse and replace thecorresponding mouse gene, in order to generate humanized mice such asmice with humanized TNF-alpha (TNF-alpha H-mice), and mice withhumanized IL-6 (IL-6 H-mice). Generation of mouse models of humandiseases or conditions are disclosed in Wu et al., “Correction of aGenetic Disease in Mouse via Use of CRISPR-Cas9,” Cell Stem Cell (2013)13(6): 659-62, and in Yang et al., “One-Step Generation of Mice CarryingReporter and Conditional Alleles by CRISPR/Cas-Mediated GenomeEngineering,” Cell (2013) 154(6): 1370-9, the disclosures of which areincorporated herein by reference in their entireties for all purposes.

In some embodiments, a transgenic animal having an immune cytokinereporter can be produced by inserting a sequence encoding the immunecytokine reporter at a desired insertion site on the chromosome of theanimal cell through the donor constructs described herein.

In some embodiments, a transgenic mouse harboring an ROSA26 locus can begenerated by inserting a sequence comprising the ROSA26 locus at adesired insertion site on the chromosome of the animal cell through thedonor constructs described herein.

Also provided are cells and genetically modified animals produced by anyone of the methods described herein.

Kits

Also provided herein are kits useful for any one of the methodsdescribed herein. For example, in some embodiments, there is provided akit for inserting a donor sequence at an insertion site on a chromosomein an eukaryotic cell, comprising: a) a sequence-specific nuclease thatcleaves the chromosome at the insertion site; b) a donor construct,wherein the donor construct comprises a 5′ homology arm, the donorsequence, and a 3′ homology arm, wherein the 5′ homology arm ishomologous to a sequence 5′ to the insertion site on the chromosome andwherein the 3′ homology arm is homologous to a sequence 3′ to theinsertion site on the chromosome; and c) an exonuclease, wherein thedonor construct is a linear nucleic acid or can be cleaved to produce alinear nucleic acid, and wherein the 5′ homology arm and the 3′ homologyarm are proximal to the 5′ and 3′ ends of the linear nucleic acid.

The kits described herein may also comprise a packaging to house thecontents of the kit. The packaging optionally provides a sterile,contaminant-free environment, and can be made of any of plastic, paper,foil, glass, and the like. In some embodiments, the packaging is a glassvial. In some embodiments, the kit further comprises an instruction forcarrying out any one of the methods described herein.

EXAMPLES

The following non-limiting examples further illustrate the compositionsand methods of the present invention. Those skilled in the art willrecognize that several embodiments are possible within the scope andspirit of this invention. The invention will now be described in greaterdetail by reference to the following non-limiting examples. Thefollowing examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope.

Example 1. The EKI System Significantly Increases Knock-in Efficiency ofEGFP-ACTB in U2OS Cells

A targeting scheme is shown in FIG. 1 for expressing an EGFP-ACTB fusionprotein. The targeting vector contains homology arms of ˜1 kb flankingthe EGFP sequence, and the sgRNA targets the ACTB allele at a positionnear the ACTB gene start codon ATG. After successful homologousrecombination, the EGFP sequence would be inserted after the start codonof the ACTB genomic locus for expression of the EGFP-ACTB fusionprotein. The sgRNA target sequence for the human ACTB gene was5′-cgcggcgatatcatcatccatgg-3′ (SEQ ID NO: 21).

The Cas9 sequence (SEQ ID NO: 22) is shown below. The bold underlinedsequence is the 3×FLAG tag sequence. The italic underlined sequences aretwo SV40 nuclear localization sequences (NLS).

SEQ ID NO: 22: 5'- ATG GACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGACGATGACGATAAG ATGGCC CCAAAGAAGAAGCGGAAGGTC GGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTGTGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATC CTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACCGAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGGCGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAGTGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGCTTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAAGAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTCTGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGAAAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATCGACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCTGCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGCCGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCTGTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGAAACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATCAGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCACCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACCATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCACCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCT CAGCTGGGAGGCGACAAAAGGCCGGCGGCCACG AAAAAGGCCGGCCAGGC AAAAAAGAAAAAGtaa-3'

The UL12 sequence (SEQ ID NO: 23) is shown below. The bold underlinedsequence is the 3×FLAG tag sequence. The italic underlined sequence is aSV40 nuclear localization sequence (NLS).

SEQ ID NO: 23:  5′-  ATG CCAAAGAAGAAGCGGAAGGTCGAGTCCACGGGAGGCCCAGCATGTCCGCCGGGACGCACCGTGACTAAGCGTTCCTGGGCCCTGGCCGAGGACACCCCTCGTGGCCCCGACAGCCCCCCCAAGCGCCCCCGCCCTAACAGTCTTCCGCTGACAACCACCTTCCGTCCCCTGCCCCCCCCACCCCAGACGACGTCAGCTGTGGACCCAAGCTCCCATTCGCCCGATAACCCCCCACGTGATCAGCACGCCACCGACACCGCAGACGAAAAGCCCCGGGCCGCGTCGCCGGCACTTTCTGACGCCTCAGGGCCTCCGACCCCAGACATTCCGCTATCTCCTGGGGGCACCCACGCCCGCGACCCGGACGCCGATCCCGACTCCCCGGACCTTGACTCTATGTGGTCGGCGTCGGTGATCCCCAACGCGCTGCCCTCCCATATACTAGCCGAGACGTTCGAGCGCCACCTGCGCGGGTTGCTGCGCGGCGTCCGCGCCCCCCTGGCCATCGGTCCCCTCTGGGCCCGCCTGGATTATCTGTGTTCCCTGGCCGTGGTCCTCGAGGAGGCGGGTATGGTGGACCGCGGACTCGGCCGGCACCTATGGCGCCTGACGCGCCGCGGGCCCCCGGCCGCCGCGGACGCCGTGGCGCCCCGGCCCCTCATGGGGTTTTACGAGGCGGCCACGCAAAACCAGGCCGACTGCCAGCTATGGGCCCTGCTCCGGCGGGGCCTCACGACCGCATCCACCCTCCGCTGGGGCCCCCAGGGTCCGTGTTTCTCGCCCCAGTGGCTGAAGCACAACGCCAGCCTGCGGCCGGATGTACAGTCTTCGGCGGTGATGTTCGGGCGGGTGAACGAGCCGACGGCCCGAAGCCTGCTGTTTCGCTACTGCGTGGGCCGCGCGGACGACGGCGGCGAGGCCGGCGCCGACACGCGGCGCTTTATCTTCCACGAACCCGGCGACCTCGCCGAAGAGAACGTGCATACGTGTGGGGTCCTCATGGACGGTCACACGGGGATGGTCGGGGCGTCCCTGGATATTCTCGTCTGTCCTCGGGACACTCACGGCTACCTGGCCCCAGTCCCCAAGACCCCCCTGGCCTTTTACGAGGTCAAATGCCGGGCCAAGTACGCTTTCGACCCCATGGACCCCAGCGACCCCACGGCCTCCGCGTACGAGGACTTGATGGCACACCGGTCCCCGGAGGCGTTCCGGGCATTTATCCGGTCGATCCCGAAGCCCAGCGTGCGATACTTCGCGCCCGGGCGCGTCCCCGGCCCGGAGGAGGCTCTCGTCACGCAAGACCAGGCCTGGTCAGAGGCCCACGCCTCGGGCGAAAAAAGGCGGTGCTCCGCCGCGGATCGGGCCTTGGTGGAGTTAAATAGCGGCGTTGTCTCGGAGGTGCTTCTGTTTGGCGCCCCCGACCTCGGACGCCAAACCATCTCCCCCGTGTCCTGGAGCTCCGGGGATCTGGTCCGCCGCGAGCCCGTCTTCGCGAACCCCCGTCACCCGAACTTTAAGCAGATCTTGGTGCAGGGCTACGTGCTCGACAGCCACTTCCCCGACTGCCCCCCCCACCCGCATCTGGTGACGTTTATCGGCAGGCACCGCACCAGCGCGGAGGAGGGCGTAACGTTCCGCCTGGAGGACGGCGCCGGGGCTCTCGGGGCCGCAGGACCCAGCAAGGCGTCCATTCTCCCGAACCAGGCCGTTCCGATCGCCCTGATCATTACCCCCGTCCGCATCGATCCGGAGATCTATAAGGCCATCCAGCGAAGCAGCCGCCTGGCGTTCGACGACACGCTCGCCGAGCTATGGGCCTCTCGTTCTCCGGGGCCCGGCCCTGCTGCTGCCGAAACAACGTCCTCATCACCGACGACGGGGAGGTCGTCTCGCG ACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAA GACGATGACGATAAG TGA-3'

The following plasmids were constructed: Cas9/sgRNA-hACTB;Cas9/sgRNA-LS14; Targeting vector TV-LS14-hACTB; and pcDNA3.1Hygro(+)—UL12.

The constructed plasmids were transfected into U2OS cells using a Neon(Invitrogen) transfection system by electroporation.

The ACTB gene encodes β-actin, which is a component of the cytoskeleton.Three days after transfection, green fluorescence indicating thefilamentous structure of the cytoskeleton was observed by fluorescentmicroscopy (FIG. 2).

Flow cytometry analysis indicated that the efficiency of conventionalCRISPR/Cas9-mediated knock-in of EGFP was only around 1.91%. Incontrast, the EKI system achieved a knock-in efficiency of 15.02% (FIG.3).

Example 2. The EKI System Significantly Increases Knock-in Efficiency ofEGFP-LMNB1 in C6 Cells

A targeting scheme is shown in FIG. 4 for expressing an EGFP-LMNB1fusion protein. The targeting vector contains homology arms of ˜1 kbflanking the EGFP sequence, and the sgRNA targets the LMNB1 allele at aposition near the LMNB1 gene start codon ATG. After successfulhomologous recombination, the EGFP sequence would be inserted after thestart codon of the LMNB1 genomic locus for expression of the EGFP-LMNB1fusion protein. The sgRNA target sequence for the human LMNB1 gene was5′-gctgtctccgccgcccgccatgg-3′ (SEQ ID NO: 24). The sgRNA target sequencefor the rat LMNB1 gene was 5′-gggggtcgcggtcgccatggcgg-3′ (SEQ ID NO:25).

The following plasmids were constructed: Cas9/sgRNA-LMNB1;Cas9/sgRNA-LS14; Targeting vector TV-LS14-LMNB1; and pcDNA3.1Hygro(+)—UL12.

The constructed plasmids were transfected into C6 cells using a Neon(Invitrogen) transfection system by electroporation.

The LMNB1 gene encodes lamin B1, which is a component of the nuclearlamina. Three days after transfection, green fluorescence indicating thenuclear membrane structure was observed by fluorescent microscopy (FIG.5).

Flow cytometry analysis indicated that the efficiency of conventionalCRISPR/Cas9-mediated knock-in of EGFP was only around 0.19%. Incontrast, the EKI system achieved a knock-in efficiency of 3.6% (FIG.6).

In another experiment, TurboGFP was used instead of EGFP for expressinga TurboGFP-LMNB1 fusion protein in C6 cells, using the targeting schemeshown in FIG. 4 (data not shown).

Example 3. The EKI System Achieves Double Knock-in of EGFP-ACTB andmCherry-LMNB1 in U2OS Cells

A targeting scheme is shown in FIG. 7 for expressing an EGFP-ACTB fusionprotein and an mCherry-LMNB1 fusion protein after knock-in of the EGFPand mCherry sequences into the endogenous ACTB and LMNB1 gene loci,respectively. The targeting vectors contain homology arms of ˜1 kbflanking the EGFP and mCherry sequences, respectively. In addition, thesgRNAs target the ACTB allele and the LMNB1 allele at positions neartheir start codons ATG. After successful homologous recombination, theEGFP sequence would be inserted after the start codon of the ACTBgenomic locus for expression of the EGFP-ACTB fusion protein, and themCherry sequence would be inserted after the start codon of the LMNB1genomic locus for expression of the mCherry-LMNB1 fusion protein.

The following plasmids were constructed: Cas9/sgRNA-ACTB;Cas9/sgRNA-LMNB1; Cas9/sgRNA-LS14; Targeting vector TV-LS14-ACTB;Targeting vector TV-LS14-LMNB1; and pcDNA3.1 Hygro(+)—UL12.

The constructed plasmids were transfected into U2OS cells using a Neon(Invitrogen) transfection system by electroporation. Three days aftertransfection, green fluorescence indicating the filamentous structure ofthe cytoskeleton and red fluorescence indicating the nuclear membranestructure were observed by fluorescent microscopy (FIG. 8).

Example 4. Using the EKI System to Efficiently Generate CD4-2A-dsRedKnock-in Rats

A targeting scheme is shown in FIG. 9 for generating CD4-2A-dsRedknock-in rats. The targeting vector contains homology arms of ˜1 kbflanking the 2A-dsRed sequence, and the sgRNA targets the endogenous ratCD4 allele at a position near the termination codon. After successfulhomologous recombination, the 2A-dsRed sequence inserted near thetermination codon of the CD4 genomic locus would be expressed as theCD4-2A-dsRed fusion protein. CD4 positive cells of the knock-in ratwould express the 2A-dsRed red fluorescent protein. The sgRNA targetsequence for the rat CD4 gene was 5′-gaaaagccacaatctcatatgagg-3′ (SEQ IDNO: 26). The sgRNA target sequence for LS14 was5′-ggtattcactcctaaagcgtcgg-3′ (SEQ ID NO: 27).

An exemplary targeting vector is shown in the 5′ to 3′ direction:

CCGACGCTTTAGGAGTGAATACC (SEQ ID NO: 28, L514sequence)-Left Homology Arm-2A-dsRed-Right Homology Arm-CCGACGCTTTAGGAGTGAATACC (SEQ ID NO: 29, LS14 sequence).

A U6-sgRNA backbone sequence (SEQ ID NO: 30) can be used. The underlinedsequence is the U6 promoter sequence, the bolded sequence is replaced bya target sequence when making a construct, and the italicized sequenceis the structural sequence of the sgRNA.

SEQ ID NO: 30: GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACC N NNNNNNNNNGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT

The T7-sgRNA backbone sequence (SEQ ID NO: 31) is shown below. Theunderlined sequence is the T7 promoter sequence, the bolded sequence isreplaced by a target sequence when making a construct, and theitalicized sequence is the structural sequence of the sgRNA.

SEQ ID NO: 31: TAATACGACTCACTATAGG NNNNNNNNNN GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAG TCGGTGCTTTT

The following plasmids were constructed: Cas9/sgRNA-CD4;Cas9/sgRNA-LS14; Targeting vector TV-LS14-CD4; pcDNA3.1 Hygro(+)—UL12;T7-Cas9; T7-sgRNA-CD4; and T7-sgRNA-LS14.

The constructed plasmids were transcribed in vitro to obtain UL12 mRNA,Cas9 mRNA, and sgRNAs for CD4 and LS14. UL12 mRNA, Cas9 mRNA, sgRNA-CD4,sgRNA-LS14, and TV-LS14-CD4 were then injected into fertilized eggs ofrats. The injected fertilized eggs were then transplanted intopseudopregnant rats.

33 rats of the F0 generation were born and genotyped. Primers LF and INRwere used in the 5′-junction PCR reaction (FIG. 9). The forward primerLF is localized distal to the left homology arm, and the reverse primerINR is localized within the 2A-dsRed region. 7 of the 33 F0 rats weretested positive for the knock-in (FIG. 10).

Primers INF and RR were used in the 3′-junction PCR reaction (FIG. 9).The forward primer INF is localized within the 2A-dsRed region, and thereverse primer RR is localized distal to the right homology arm. 7 ofthe 33 F0 rats were tested positive for the knock-in (FIG. 11).

Thus, the 5′-junction PCR reaction and the 3′-junction PCR reactionyielded the same results, and F0 rats numbered #6, #7, #14, #17, #22,#23, and #29 were tested positive for the knock-in by both PCR assays.The positive rate is 7/33 (21.2%).

FIG. 12 shows southern blot results for the knock-in rat, furtherindicating insertion of the donor sequence at the predetermined sites.The two F1 rats (#19 and #21) tested with southern blots were theoffspring of the #22 F0 rat in FIG. 10 and FIG. 11.

Example 5. Using the EKI System to Generate Knock-in of TH-GFP in H9Cells

This example illustrates the generation of a TH-GFP knock-in in H9 humanembryonic stem cells using the EKI system.

The human TH gene encodes tyrosine hydroxylase (also known as tyrosine3-monooxygenase or tyrosinase), an enzyme that catalyzes the amino acidL-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) conversion. L-DOPAis a precursor for Dopamine. TH is expressed in the central nervoussystem, peripheral sympathetic neurons and the adrenal medulla, and isused as a dopaminergic neuron marker.

A targeting scheme is shown in FIG. 13 for generating a TH-GFP knock-inH9 cell line. The targeting vector contains homology arms of ˜1 kbupstream of the F2A-GFP cassette and downstream of the PGK-EM7-Neo-SV40polyA cassette. The sgRNA targets the TH allele at a position near itsstop codon. After successful homologous recombination, the F2A-GFPcassette and the PGK-EM7-Neo-SV40 polyA cassette would be placedimmediately before the stop codon and after the 3′UTR of the TH gene,respectively, for the expression of a TH-GFP fusion protein. The sgRNAtarget sequence for the human TH gene was 5′-ggacgccgtgcacctagccaatgg-3′ (SEQ ID NO: 44).

The following plasmids were constructed: Cas9/sgRNA-TH; Cas9/sgRNA-LS14;Targeting vector TV-LS14-TH; and pcDNA3.1 Neo(+)—UL12.

The constructed plasmids were transfected into H9 cells using a Neon(Invitrogen) transfection system by electroporation. 2×10⁶ H9 cells, and2.5 μg each of Cas9/sgRNA-TH, Cas9/sgRNA-LS14, TV-LS14-TH, and pcDNA3.1Neo(+)—UL12 were used. Drug-resistant colonies were picked and expandedafter 7-10 days of G418 selection.

The green fluorescence from GFP can serve as a marker of the TH geneactivity, which was found to express when H9 cells differentiated intodopaminergic neurons (see FIG. 14). This H9-TH-GFP cell line can be usedto support research in many areas including dopaminergic neurondifferentiation from human embryonic stem cells.

The knock-in of TH-GFP in H9 cells was genotyped in three independentcell lines using primers listed in Table 1, reaction components listedin Table 2, and the PCR cycling condition listed in Table 3.

TABLE 1 Primers used for H9-TH-GFP genotyping PCR Tm  Product ProductPrimer Sequence (5'-3') (° C.) size (bp) 5'PCR hTH-5'-FAGTGGAGTCAGTGATGCCATTGGCCTC (SEQ ID NO: 32) 65 1362 hTH-5'-RGCCTTTGGTGCTCTTCATCTTGTTGG (SEQ ID NO: 33) 61 3'PCR hTH-3'-FTACCCGTGATATTGCTGAAGAGCTTG (SEQ ID NO: 34) 60 1751 hTH-3'-RTTTGGTAGTGGGCACCAGCTATCTG (SEQ ID NO: 35) 61

TABLE 2 PCR reaction components H₂O 17.75 μl  KOD buffer (10x)   3 μldNTP (2 mM)   3 μl DMSO (0.5%)  1.5 μl MgSO₄ (25 mM)  1.5 μl ForwardPrimer (10 μM) 0.75 μl Reverse Primeer (10 μM) 0.75 μl Genomic DNA(100~200 ng/μl)   1 μl KOD-plus 0.75 μl Total  30 μl

TABLE 3 PCR cycling condition 94° C. 5 min 94° C. 30 sec 67° C.(−0.7°C./cycle) 30 sec {close oversize brace} 15 cycles 68° C. 1 min/kb 94° C.30 sec 57° C. 30 sec {close oversize brace} 25 cycles 68° C. 1 min/kb68° C. 10 min  4° C. Hold

Primers TH-5′-F and TH-5′-R were used in the 5′-junction PCR reaction(FIG. 15). The forward primer TH-5′-F is localized distal to the lefthomology arm, and the reverse primer TH-5′-R is localized at the F2A-GFPcassette. All three cell lines were tested positive for the knock-in(FIG. 16A).

Primers TH-3′-F and TH-3′-R were used in the 3′-junction PCR reaction(FIG. 15). The forward primer TH-3′-F is localized at thePGK-EM7-Neo-SV40 polyA cassette, and the reverse primer TH-3′-R islocalized distal to the right homology arm. All three cell lines weretested positive for the knock-in (FIG. 16B).

Thus, 5′-junction PCR and 3′-junction PCR yielded the same results, andcell lines numbered #1, #2 and #3 were all tested positive for theknock-in by both PCR assays.

FIG. 17 shows the sequencing results of the PCR products from cell line#1, which confirmed that the cell line was correctly targeted at the THlocus.

FIG. 18 shows that the tested cell line #1 has normal human karyotype.

Example 6. Using the EKI System to Generate Knock-in of OCT4-EGFP in H9Cells

This example illustrates the generation of an OCT4-EGFP knock-in in H9human embryonic stem cells using the EKI system.

OCT4 (octamer-binding transcription factor 4; also known as POU5F1:POUdomain, class 5, transcription factor 1), encoded by the POU5F1 gene inhuman, is a transcription factor that binds to the octamer motif(5′-ATTTGCAT-3′). It plays a critical role in embryonic development andstem cell self-renewal and pluripotency. OCT4 is expressed in humanembryonic stem cells, germ cells, and adult stem cells. Aberrantexpression of this gene in adult cells is associated with tumorigenesis.

A targeting scheme is shown in FIG. 19 for generating an OCT4-EGFPknock-in H9 cell line. The targeting vector contains homology arms of ˜1kb flanking the EGFP-F2A-Puro-SV40-polyA signal sequence cassette. ThesgRNA targets the OCT4 allele at a position near its stop codon. Aftersuccessful homologous recombination, the EGFP-F2A-Puro-SV40-polyA signalsequence cassette would be placed immediately before the stop codon ofthe OCT4 gene, for the expression of an OCT4-EGFP fusion protein. ThesgRNA target sequence for the human OCT4 gene was5′-tctcccatgcattcaaactgagg-3′ (SEQ ID NO: 45).

The following plasmids were constructed: Cas9/sgRNA-OCT4;Cas9/sgRNA-LS14; Targeting vector TV-LS14-OCT4; and pcDNA3.1Puro(+)—UL12.

The constructed plasmids were transfected into H9 cells using a Neon(Invitrogen) transfection system by electroporation. 2×10⁶ H9 cells, and2.5 μg each of Cas9/sgRNA-OCT4, Cas9/sgRNA-LS14, TV-LS14-OCT4, andpcDNA3.1 Puro(+)—UL12 were used. Drug-resistant colonies were picked andexpanded after 7-10 days of Puromycin selection.

The green fluorescence from EGFP can serve as a marker of the OCT4 geneactivity, which was found to express in pluripotent stem cells (see FIG.20). This H9-OCT4-EGFP cell line can be used to support research in manyareas including reprogramming and human embryonic stem cell self-renewaland differentiation.

The knock-in of OCT4-EGFP in H9 cells was genotyped in 15 independentcell lines using primers listed in Table 4, reaction components listedin Table 2, and the PCR cycling condition listed in Table 3.

TABLE 4 Primers used for H9-OCT4-EGFP genotyping PCR Product ProductPrimer Sequence (5'-3') size (bp) 5' PCR OCT4-5'-FGGTATTCAGCCAAACGACCATCTGCCG (SEQ ID NO: 36) 1344 OCT4-5'-RAGTCGTGCTGCTTCATGTGGTCG (SEQ ID NO: 37) 3' PCR OCT4-3'-FTGACACGTGCTACGAGATTTCGATTC (SEQ ID NO: 38) 1354 OCT4-3'-RACAGGCTTCACCTGTACTGTCAGGGCA (SEQ ID NO: 39) Full Length OCT4-5'-FGGTATTCAGCCAAACGACCATCTGCCG (SEQ ID NO: 36) 3831 OCT4-3'-RACAGGCTTCACCTGTACTGTCAGGGCA (SEQ ID NO: 39)

Primers OCT4-5′-F and OCT4-5′-R were used in the 5′-junction PCRreaction (FIG. 21). The forward primer OCT4-5′-F is localized distal tothe left homology arm, and the reverse primer OCT4-5′-R is localized atthe EGFP-F2A-Puro-SV40-polyA signal sequence cassette. All 15 cell lineswere tested positive for the knock-in (FIG. 22A).

Primers OCT4-3′-F and OCT4-3′-R were used in the 3′-junction PCRreaction (FIG. 21). The forward primer OCT4-3′-F is localized at theEGFP-F2A-Puro-SV40-polyA signal sequence cassette, and the reverseprimer OCT4-3′-R is localized distal to the right homology arm. Celllines numbered #1, #2, #4, #6, #7, #10, #11 and #12 were tested positivefor the knock-in (FIG. 22B).

Full length PCR reaction was further tested with primers OCT4-5′-F andOCT4-3′-R (FIG. 21). Cell lines numbered #1, #3, #4, #5, #6, #7, #10,#11 and #13 were tested positive for the knock-in (FIG. 22C).

FIG. 23 shows the sequencing results of the PCR products from cell line#6, which confirmed that the cell line was correctly targeted at theOCT4 locus.

FIG. 24 shows that the tested cell line #6 has normal human karyotype.

REFERENCES

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What is claimed is:
 1. A method of inserting a donor sequence at a predetermined insertion site on a chromosome in an eukaryotic cell, comprising: a) introducing into the cell a first sequence-specific nuclease that cleaves the chromosome at the insertion site; b) introducing into the cell a circular donor construct; c) introducing into the cell an exonuclease; and d) introducing into the cell a second sequence-specific nuclease; wherein the circular donor construct is cleaved within the cell by the second sequence-specific nuclease to produce a linear nucleic acid, wherein the linear nucleic acid comprises a 5′ homology arm, the donor sequence, and a 3′ homology arm, wherein the 5′ homology arm is homologous to a sequence upstream of the nuclease cleavage site on the chromosome and wherein the 3′ homology arm is homologous to a sequence downstream of the nuclease cleavage site on the chromosome; wherein the 5′ homology arm and the 3′ homology arm are proximal to the 5′ and 3′ ends of the linear nucleic acid, respectively, wherein the circular donor construct comprises a 5′ flanking sequence upstream of the 5′ homology arm and a 3′ flanking sequence downstream of the 3′ homology arm, wherein the second sequence-specific nuclease cleaves the circular donor construct at both of the 5′ flanking sequence and the 3′ flanking sequence, thereby producing the linear nucleic acid, wherein the donor sequence is inserted into the chromosome at the insertion site through homologous recombination, and wherein the 5′ flanking sequence and the 3′ flanking sequence each comprises a sequence selected from the group consisting of SEQ ID NOs: 3-20.
 2. The method of claim 1, wherein the first sequence-specific nuclease is zinc finger nuclease (ZFN).
 3. The method of claim 1, wherein the first sequence-specific nuclease is a transcription activator-like effector nuclease (TALEN).
 4. The method of claim 1, wherein the first sequence-specific nuclease is an RNA-guided nuclease.
 5. The method of claim 4, wherein the RNA-guided nuclease is Cas.
 6. The method of claim 5, wherein the RNA-guided nuclease is Cas9.
 7. The method of claim 4, further comprising introducing into the cell a first guide RNA recognizing the insertion site.
 8. The method of claim 1, wherein the first sequence-specific nuclease is introduced into the cell as a protein, mRNA, or cDNA.
 9. The method of claim 1, wherein the 5′ homology arm and the 3′ homology arm are at least 200 bp.
 10. The method of claim 1, wherein the exonuclease is a 5′ to 3′ exonuclease.
 11. The method of claim 1, wherein the second sequence-specific nuclease is an RNA-guided nuclease, and wherein the method further comprises introducing into the cell a second guide RNA recognizing both of the flanking sequences.
 12. A method of generating a genetically modified non-human animal comprising a donor sequence inserted at a predetermined insertion site on the chromosome of the non-human animal, comprising introducing the cell generated by the method of claim 1 into a carrier non-human animal to produce the genetically modified non-human animal.
 13. The method of claim 12, wherein the cell is a zygote or a pluripotent stem cell.
 14. A kit for inserting a donor sequence at an insertion site on a chromosome in an eukaryotic cell, comprising: a) a first sequence-specific nuclease that cleaves the chromosome at the insertion site; b) a circular donor construct; c) an exonuclease; and d) a second sequence-specific nuclease; wherein the circular donor construct can be cleaved within the eukaryotic cell by the second sequence-specific nuclease to produce a linear nucleic acid, wherein the linear nucleic acid comprises a 5′ homology arm, the donor sequence, and a 3′ homology arm, wherein the 5′ homology arm is homologous to a sequence upstream of the nuclease cleavage site on the chromosome and wherein the 3′ homology arm is homologous to a sequence downstream of the nuclease cleavage site on the chromosome; wherein the circular donor construct comprises a 5′ flanking sequence upstream of the 5′ homology arm and a 3′ flanking sequence downstream of the 3′ homology arm; wherein the 5′ homology arm and the 3′ homology arm are proximal to the 5′ and 3′ ends of the linear nucleic acid, respectively; wherein the second sequence-specific nuclease is capable of cleaving the circular donor construct at both of the 5′ flanking sequence and the 3′ flanking sequence, thereby producing the linear nucleic acid; and wherein the 5′ flanking sequence and the 3′ flanking sequence each comprises a sequence selected from the group consisting of SEQ ID NOs: 3-20.
 15. The method of claim 1, wherein the 5′ flanking sequence or the 3′ flanking sequence is about 1 to about 500 bp.
 16. The method of claim 10, wherein the exonuclease is a herpes simplex virus type 1 (HSV-1) exonuclease.
 17. The method of claim 16, wherein the exonuclease is UL12.
 18. The method of claim 1, wherein the first sequence-specific nuclease and the second sequence-specific nuclease are both ZFNs, both TALENs, or both RNA-guide nucleases.
 19. A method of inserting a donor sequence at a predetermined insertion site on a chromosome in an eukaryotic cell, comprising: a) introducing into the cell a sequence-specific RNA-guided nuclease; b) introducing into the cell a circular donor construct; c) introducing into the cell a first guide RNA recognizing the insertion site; d) introducing into the cell a second guide RNA recognizing a cleavage site on the circular donor construct; and e) introducing into the cell an exonuclease; wherein upon cleavage within the cell by the sequence-specific RNA-guided nuclease the circular donor construct produces a linear nucleic acid comprising a 5′ homology arm, the donor sequence, and a 3′ homology arm, wherein the 5′ homology arm is homologous to a sequence upstream of the nuclease cleavage site on the chromosome and wherein the 3′ homology arm is homologous to a sequence downstream of the nuclease cleavage site on the chromosome; wherein the 5′ homology arm and the 3′ homology arm are proximal to the 5′ and 3′ ends of the linear nucleic acid, respectively; wherein the circular donor construct comprises a 5′ flanking sequence upstream of the 5′ homology arm and a 3′ flanking sequence downstream of the 3′ homology arm; wherein the sequence-specific RNA-guided nuclease cleaves the circular donor construct at both of the 5′ flanking sequence and the 3′ flanking sequence, thereby producing the linear nucleic acid, wherein the donor sequence is inserted into the chromosome at the insertion site through homologous recombination, and wherein the 5′ flanking sequence and the 3′ flanking sequence each comprises a sequence selected from the group consisting of SEQ ID NOs: 3-20. 